Probiotic, lactic acid-producing bacteria and uses thereof

ABSTRACT

The present invention discloses compositions and methodologies for the utilization of probiotic organisms in therapeutic compositions. More specifically, the present invention relates to the utilization of one or more species or strains of lactic acid-producing bacteria, preferably strains of  Bacillus coagulans , for the control of gastrointestinal tract pathogens, including antibiotic-resistant gastrointestinal tract pathogens, and their associated diseases by both a reduction in the rate of colonization and the severity of the deleterious physiological effects of the colonization of the antibiotic-resistant pathogen. In addition, the present invention relates to the utilization of therapeutic compounds comprised of lactic acid-producing bacteria and anti-microbial agents such as antibiotics, anti-fungal compounds, anti-yeast compounds, or anti-viral compounds. The present invention also discloses methodologies for: (i) the selective breeding and isolation of probiotic, lactic acid-producing bacterial strains which possess resistance or markedly decreased sensitivity to anti-microbial agents (e.g., antibiotics, anti-fungal agents, anti-yeast agents, and anti-viral agents); and (ii) treating or preventing bacteria-mediated infections of the gastrointestinal tract by use of the aforementioned probiotic bacterial strains with or without the concomitant administration of antibiotics. While the primary focus is on the treatment of gastrointestinal tract infections, the therapeutic compositions of the present invention may also be administered to buccal, vaginal, optic, and like physiological locations.

FIELD OF THE INVENTION

[0001] The present invention relates to methods and compositions for theutilization of probiotic organisms in therapeutic compositions. Morespecifically, the present invention relates to the utilization of one ormore species or strains of lactic acid-producing bacteria, preferablystrains of Bacillus coagulans, for the control of gastrointestinal tractpathogens, including antibiotic-resistant gastrointestinal tractpathogens, and their associated diseases by both a reduction in the rateof colonization and the severity of the deleterious physiologicaleffects of the colonization of the antibiotic-resistant pathogen. Inaddition, the present invention relates to the utilization oftherapeutic compounds comprised of lactic acid-producing bacteria andanti-microbial agents such as antibiotics, anti-fungal compounds,anti-yeast compounds, or anti-viral compounds. In addition, the presentinvention relates to the use of lactic acid-producing bacteria inanimals to mitigate gastrointestinal tract pathogens.

BACKGROUND OF THE INVENTION

[0002] 1. Probiotic Microorganisms

[0003] The gastrointestinal microflora has been shown to play a numberof vital roles in maintaining gastrointestinal tract function andoverall physiological health. For example, the growth and metabolism ofthe many individual bacterial species inhabiting the gastrointestinaltract depend primarily upon the substrates available to them, most ofwhich are derived from the diet. See e.g., Gibson G. R. et al., 1995.Gastroenterology 106: 975-982; Christi, S. U. et al., 1992. Gut 33:1234-1238. These finding have led to attempts to modify the structureand metabolic activities of the community through diet, primarily withprobiotics which are live microbial food supplements. The best knownprobiotics are the lactic acid-producing bacteria (i.e., Lactobacilli)and Bifidobacteria, which are widely utilized in yogurts and other dairyproducts. These probiotic organisms are non-pathogenic andnon-toxigenic, retain viability during storage, and survive passagethrough the stomach and small intestine. Since probiotics do notpermanently colonize the host, they need to be ingested regularly forany health promoting properties to persist. Commercial probioticpreparations are generally comprised of mixtures of Lactobacilli andBifidobacteria, although yeast such as Saccharomyces have also beenutilized.

[0004] Probiotic preparations were initially systematically evaluatedfor their effect on health and longevity in the early-1900's (see e.g.,Metchinikoff, E., Prolongation of Life, Willaim Heinermann, London1910), although their utilization has been markedly limited since theadvent of antibiotics in the 1950's to treat pathological microbes. Seee.g., Winberg, et al, 1993. Pediatr. Nephrol. 7: 509-514; Malin et al,Ann. Nutr. Metab. 40: 137-145; and U.S. Pat. No. 5,176,911. Similarly,lactic acid-producing bacteria (e.g., Bacillus, Lactobacillus andStreptococcus species) have been utilized as food additives and therehave been some claims that they provide nutritional and/or therapeuticvalue. See e.g., Gorbach, 1990. Ann. Med. 22: 37-41; Reid et al, 1990.Clin. Microbiol. Rev. 3: 335-344.

[0005] Therefore, probiotic microorganisms are those which confer abenefit when grow in a particular environment, often by inhibiting thegrowth of other biological organisms in the same environment. Examplesof probiotic organisms include bacteria and bacteriophages which possessthe ability to grow within the gastrointestinal tract, at leasttemporarily, to displace or destroy pathogenic organisms, as well asproviding other benefits to the host. See e.g., Salminen et al, 1996.Antonie Van Leeuwenhoek 70: 347-358; Elmer et al. 1996. JAMA 275:870-876; Rafter, 1995. Scand. J. Gastroenterol. 30: 497-502; Perdigon etal, 1995. J. Dairy Sci. 78: 1597-1606; Gandi, Townsend Lett. Doctors &Patients, pp. 108-110, January 1994; Lidbeck et al, 1992. Eur. J. CancerPrev. 1: 341-353.

[0006] The majority of previous studies on probiosis have beenobservational rather than mechanistic in nature, and thus the processesresponsible for many probiotic phenomena have yet to be quantitativelyelucidated. Some probiotics are members of the normal colonic microfloraand are not viewed as being overtly pathogenic. However, these organismshave occasionally caused infections (e.g., bacteremia) in individualswho are, for example, immunocompromised. See e.g., Sussman, J. et al.,1986. Rev Infect. Dis. 8: 771-776; Hata, D. et al., 1988. Pediatr.Infect. Dis. 7: 669-671.

[0007] While the attachment of probiotics to the gastrointestinalepithelium is an important determinant of their ability to modify hostimmune reactivity, this is not a universal property of Lactobacilli orBifidobacteria, nor is it essential for successful probiosis. See e.g.,Fuller, R., 1989. J. Appl. Bacteriol. 66: 365-378. For example,adherence of Lactobacillus acidophilus and some Bifidobacteria to humanenterocyte-like CACO-2 cells has been demonstrated to prevent binding ofenterotoxigenic and enteropathogenic Escherichia coli, as well asSalmonella typhimurium and Yersinia pseudotuberculosis. See e.g.,Bernet, M. F. et al., 1994. Gut 35: 483-489; Bernet, M. F. et al., 1993.Appl. Environ. Microbiol. 59: 4121-4128.

[0008] While the gastrointestinal microflora presents a microbial-basedbarrier to invading organisms, pathogens often become established whenthe integrity of the microbiota is impaired through stress, illness,antibiotic treatment, changes in diet, or physiological alterationswithin the G.I. tract. For example, Bifidobacteria are known to beinvolved in resisting the colonization of pathogens in the largeintestine. See e.g., Yamazaki, S. et al., 1982. Bifidobacteria andMicroflora 1: 55-60. Similarly, the administration of Bifidobacteriabreve to children with gastroenteritis eradicated the causativepathogenic bacteria (i.e., Campylobacter jejuni) from their stools (seee.g., Tojo, M., 1987. Acta Pediatr. Jpn. 29: 160-167) andsupplementation of infant formula milk with Bifidobacteria bifidum andStreptococcus thermophilus was found to reduce rotavirus shedding andepisodes of diarrhea in children who were hospitalized (see e.g.,Saavedra, J. M., 1994. The Lancet 344: 1046-109.

[0009] In addition, some lactic acid producing bacteria also producebacteriocins which are inhibitory metabolites which are responsible forthe bacteria's anti-microbial effects. See e.g., Klaenhammer, 1993. FEMSMicrobiol. Rev. 12: 39-85; Barefoot et al., 1993. J Diary Sci. 76:2366-2379. For example, selected Lactobacillus strains which produceantibiotics have been demonstrated as effective for the treatment ofinfections, sinusitis, hemorrhoids, dental inflammations, and variousother inflammatory conditions. See e.g., U.S. Pat. No. 5,439,995.Additionally, Lactobacillus reuteri has been shown to produceantibiotics which possess anti-microbial activity against Gram negativeand Gram positive bacteria, yeast, and various protozoan. See e.g., U.S.Pat. Nos. 5,413,960 and 5,439,678.

[0010] Probiotics have also been shown to possess anti-mutagenicproperties. For example, Gram positive and Gram negative bacteria havebeen demonstrated to bind mutagenic pyrolysates which are producedduring cooking at a high temperature. Studies performed with lacticacid-producing bacteria has shown that these bacteria may be eitherliving or dead, due to the fact that the process occurs by adsorption ofmutagenic pyrolysates to the carbohydrate polymers present in thebacterial cell wall. See e.g., Zang, X. B. et al., 1990. J. Dairy Sci.73: 2702-2710. Lactobacilli have also been shown to degrade carcinogens(e.g., N-nitrosamines), which may serve an important role if the processis subsequently found to occur at the level of the mucosal surface. Seee.g., Rowland, I. R. and Grasso, P., Appl. Microbiol. 29: 7-12.Additionally, the co-administration of lactulose and Bifidobacterialongum to rats injected with the carcinogen azoxymethane wasdemonstrated to reduce intestinal aberrant crypt foci, which aregenerally considered to be pre-neoplastic markers. See e.g., Challa, A.et al., 1997. Carcinogenesis 18: 5175-21. Purified cell walls ofBifidobacteria may also possess anti-tumorigenic activities in that thecell wall of Bifidobacteria infantis induces the activation ofphagocytes to destroy growing tumor cells. See e.g., Sekine, K. et al.,1994. Bifidobacteria and Microflora 13: 65-77. Bifidobacteria probioticshave also been shown to reduce colon carcinogenesis induced by1,2-dimethylhydrazine in mice when concomitantly administered withfructo-oligosaccharides(FOS; see e.g., Koo, M. B., and Rao, A. V., 1991.Nutrit. Rev. 51: 137-146), as well as inhibiting liver and mammarytumors in rats (see e.g., Reddy, B. S., and Rivenson, A., 1993. CancerRes. 53: 3914-3918).

[0011] It has also been-demonstrated that the microbiota of thegastrointestinal tract affects both mucosal and systemic immunity withinthe host. See e.g., Famularo, G. et al., Stimulation of Immunity byProbiotics. In: Probiotics: Therapeutic and Other Beneficial Effects.pg. 133-161. (Fuller, R., ed. Chapman and Hall, 1997). The intestinalepithelial cells, blood leukocytes, B- and T-lymphocytes, and accessorycells of the immune system have all been implicated in theaforementioned immunity. See e.g., Schiffrin, E. J. et al., 1997. Am. JClin. Nutr. 66(suppl): 5-20S. Other bacterial metabolic products whichpossess immunomodulatory properties include: endotoxiclipopolysaccharide, peptidoglycans, and lipoteichoic acids. See e.g.,Standiford, T. K., 1994. Infect. Linmun. 62: 119-125. Accordingly,probiotic organisms are thought to interact with the immune system atmany levels including, but not limited to: cytokine production,mononuclear cell proliferation, macrophage phagocytosis and killing,modulation of autoimmunity, immunity to bacterial and protozoanpathogens, and the like. See e.g., Matsumara, K. et al., 1992. AnimalSci. Technol. (Jpn) 63: 1157-1159; Solis-Pereyra, B. and Lemmonier, D.,1993. Nutr. Res. 13: 1127-1140. Lactobacillus strains have also beenfound to markedly effect changes in inflammatory and immunologicalresponses including, but not limited to, a reduction in colonicinflammatory infiltration without eliciting a similar reduction in thenumbers of B- and T-lymphocytes. See e.g., De Simone, C. et al., 1992.Immunopharmacol. Immunotoxicol. 14: 331-340.

[0012] 2. Gastrointestinal Effects of Antibiotic Administration

[0013] Antibiotics are widely used to control pathogenic microorganismsin both humans and animals. Unfortunately, the widespread use ofanti-microbial agents, especially broad spectrum antibiotics, hasresulted in a number of serious clinical consequences. For example,antibiotics often kill beneficial, non-pathogenic microorganisms (i.e.,flora) within the gastrointestinal tract which contribute to digestivefunction and health. Accordingly, relapse (the return of infections andtheir associated symptoms) and secondary opportunistic infections oftenresult from the depletion of lactic acid-producing and other beneficialflora within the gastrointestinal tract.

[0014] Unfortunately, most, if not all, lactic acid-producing orprobiotic bacteria are extremely sensitive to common antibioticcompounds Accordingly, during a normal course of antibiotic therapy,many individuals develop a number of deleterious physiologicalside-effects including: diarrhea, intestinal cramping, and sometimesconstipation. These side-effects are primarily due to the non-selectiveaction of antibiotics, as antibiotics do not possess the ability todiscriminate between beneficial, non-pathogenic and pathogenic bacteria,both bacterial types are killed by these agents. Thus, individualstaking antibiotics offer suffer from gastrointestinal problems as aresult of the beneficial microorganisms (i.e., intestinal flora), whichnormally colonize the gastrointestinal tract, being killed or severelyattenuated. The resulting change in the composition of the intestinalflora can result in vitamin deficiencies when the vitamin-producingintestinal bacteria are killed, diarrhea and dehydration and, moreseriously, illness should a pathogenic organism overgrow and replace theremaining beneficial gastrointestinal bacteria.

[0015] Another deleterious result of indiscriminate use ofanti-microbial agents is the generation of multiple antibiotic-resistantpathogens. See e.g., Mitchell, P. 1998. The Lancet 352: 462-463;Shannon, K., 1998. Lancet 352: 490-491. The initial reports ofmeticillin-resistant Staphylococcus aurous (MRSA) infections have beenover-shadowed by the more recent outbreaks of vancomycin-resistantEnterococci (VRE). The development of such resistance has led tonumerous reports of systemic infections which remained untreatable withconventional antibiotic therapies. Recently, a vancomycin-(generallyregarded as an antibiotic of “last resort”) resistant strain ofStaphylococcus aurous was responsible for over 50 deaths in a singleAustralian hospital. See e.g., Shannon, K., 1998. Lancet 352: 490-491.

[0016] Enterococci are currently a major nosocomial pathogen and arelikely to remain as such for a long period of time. Enterococci, as wellas other microbes, obtain antibiotic resistance genes in severaldifferent ways. For example, Enterococci emit pheromones which causethem to become “sticky” and aggregate, thus facilitating the exchange ofgenetic material, such as plasmids (autonomously replicating, circularDNA which often carry the antibiotic resistance genes). In addition,some Enterococci also possess “conjugative transposons” which are DNAsequences that allow them to directly transfer resistance genes withoutplasmid intermediary. It is believed that penicillin resistance has beenconferred from Enterococci to Streptococci to Staphylococci through thislater mechanism.

[0017] Since 1989, a rapid increase in the incidence of infection andcolonization with vancomycin-resistant Enterococci (VRE) has beenreported by numerous hospitals within the United States. This increaseposes significant problems, including: (i) the lack of availableanti-microbial therapy for VRE infections, due to the fact that most VREare also resistant to the drugs which were previously used to treat suchinfections (e.g., Aminoglycosides and Ampicillin); and (ii) thepossibility that the vancomycin-resistant genes present in VRE can betransferred to other gram-positive microorganisms (e.g., Staphylococcusaureus).

[0018] An increased risk for VRE infection and colonization has alsobeen associated with previous vancomycin and/or multi-anti-microbialtherapy, severe underlying disease or immunosuppression, andintra-abdominal surgery. Because Enterococci can be found within thenormal gastrointestinal and female genital tracts, most enterococcalinfections have been attributed to endogenous sources within theindividual patient. However, recent reports of outbreaks and endemicinfections caused by Enterococci, including VRE, have indicated thatpatient-to-patient transmission of the microorganisms can occur througheither direct contact or through indirect contact via (i) the hands ofpersonnel; or (ii) contaminated patient-care equipment or environmentalsurfaces.

[0019] Accordingly, there remains a need for a highly efficaciousbiorational therapy which functions to mitigate the deleteriousphysiological effects of digestive pathogens, includingantibiotic-resistant gastrointestinal tract pathogens, in both humansand animals, by the colonization (or re-colonization) of thegastrointestinal tract with probiotic microorganisms, following theadministration of antibiotics, anti-fungal, anti-viral, and similaragents. Additionally, a need as remains for the development of a highlyefficacious biorational therapy which functions to mitigateantibiotic-resistant digestive pathogens, in both humans and animals, bythe colonization (or re-colonization) of the gastrointestinal tract withprobiotic microorganisms, following the administration of antibiotics,anti-fungal, anti-viral, and similar agents, by functioning to reduceboth the colonization rate and the potential physiologically deleteriouseffects due to the colonization of antibiotic-resistant digestivepathogens.

SUMMARY OF THE INVENTION

[0020] The present invention discloses methodologies for the selectivebreeding and isolation of antibiotic-resistant, lactic acid-producingbacterial strains for utilization in various types of therapeuticapplications. For example, in one specific embodiment, these lacticacid-producing bacteria are co-administered with one or moreanti-microbial compounds (e.g., antibiotics, anti-mycotic compounds,anti-viral compounds, and the like). It should be noted that, in mostclinical and scientific fields, the production or evolution ofantibiotic resistant microorganisms is an undesirable consequence ofunnecessary issue and/or improper use of antibiotics compounds. However,the present invention serves to constructively produce bacteria thatpossess resistance to a single, as opposed to multiple, antibiotics.

[0021] In another related aspect, the present invention disclosescompositions and methodologies for the utilization of these compositionscomprising non-pathogenic, probiotic lactic acid-producing bacteriawhich are used to mitigate the deleterious physiological effects ofgastrointestinal tract pathogens, including antibiotic-resistantgastrointestinal tract pathogens, in both humans and animals, by thecolonization (or more-correctly, re-colonization) of thegastrointestinal tract with probiotic microorganisms, following theadministration of antibiotics, anti-fungal, anti-viral, and similaragents.

[0022] Additionally, the present invention relates to the use of lacticacid-producing bacteria to mitigate the effects of parasites andpathogens in animals.

[0023] 1. Co-Administration of Probiotic bacterial with Anti-MicrobialCompounds

[0024] It has been demonstrated that common and antibiotic resistantdigestive pathogens can be controlled with the utilization of particularprobiotic organisms that have been identified for their ability toremain viable in the gastrointestinal tract during antibiotic therapy.However, it should be noted that, prior to the disclosure of the presentinvention, most strains of probiotic bacteria (e.g., Lactobacillus,Bifidiobacterium, and Bacillus) were found to be sensitive to themajority of antibiotics, hence they were not particularly suitable forco-administration with broad-spectrum antibiotics.

[0025] Accordingly, in the present invention, strains of Bacilluscoagulans were isolated and identified for their ability to remainviable when exposed to typical therapeutic concentrations of antibioticsthat are commonly used to mitigate digestive pathogens. These newBacillus variants disclosed herein may be administered prior to,concomitantly with, or subsequent to the administration of antibiotics.In a preferred embodiment, these Bacillus strains are co-administered incombination with the selected antibiotic which they are resistant to.

[0026] One probiotic bacterial strain disclosed by the present inventionis Bacillus coagulans GB-M—a new variant or mutant of Bacillus coagulansATCC No. 31284. Bacillus coagulans GB-M has been demonstrated to beresistant to Macrolide antibiotics such as Azithromycin, Erythromycinand other similar antibiotic compounds. The advantages of using abiological in combination with a chemical antibiotic or the concurrentuse of a biological with a chemical serves to address the many hazardsand side effects of antibiotic therapy. In addition, the use of theseaforementioned variants, as well as other lactic acid-producingbiorationals, in combination with chemotherapy drugs and anti-fungalwould be of great benefit to those taking these compounds, due to thefact that these individuals, more often than not, suffer from sideeffects which are a direct result of depleted “normal” gastrointestinalflora.

[0027] In addition to the aforementioned aspects of the presentinvention, the utilization of bifidogenic oligosaccharides (e.g.,fructo-oligosaccharides (FOS)) are beneficial to facilitate there-establishment and proliferation of other beneficial lacticacid-producing bacteria and to further promote gastrointestinalmicrobial biodiversity. In one embodiment of the present invention, acomposition comprising an isolated and specific antibiotic resistantBacillus coagulans strain in combination with an effective amount of afructo-oligosaccharide (FOS) in a pharmaceutically acceptable carriersuitable for administration to the gastrointestinal track of a human oranimal is disclosed. In preferred embodiments of the present invention,the Bacillus coagulans strain is included in the composition in the formof spores, a dried cell mass, in the form of a flowable concentrate, orin the form of a stabilized gel or paste.

[0028] In another embodiment of the present invention, the Bacilluscoagulans strain is combined with a therapeutically-effective dose of anantibiotic. In preferred embodiments of the present invention, theBacillus coagulans strain is combined with a therapeutic concentrationof antibiotic including, but not limited to: Gentamicin; Vancomycin;Oxacillin; Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-sulfamethoxasole; a member of the Cephlosporin antibioticfamily (e.g., Cefaclor, Cefadroxil, Cefixime, Cefprozil, Ceftriaxone,Cefuroxime, Cephalexin, Loracarbef, and the like); a member of thePenicillin family of antibiotics (e.g., Ampicillin,Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin. Penicillin VK, andthe like); with a member of the Fluoroquinolone family of antibiotics(e.g., Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); or amember of the Macrolide antibiotic family (e.g., Azithromycin,Erythromycin, and the like).

[0029] Similarly, a therapeutically-effective concentration of ananti-fungal agent may also be utilized. Such anti-fungal agents include,but are not limited to: Clotrimazole, Fluconazole, Itraconazole,Ketoconazole, Miconazole, Nystatin, Terbinafine, Terconazole, andTioconazole.

[0030] The aforementioned embodiment involves selectively-culturing theprobiotic bacteria (which may initially be sensitive to the antibioticof choice) in gradually increasing concentrations of antibiotic in orderto facilitate the development of decreased antibiotic sensitivity or,preferably, total antibiotic resistance. It should be noted that this isthe most preferred embodiment of the present invention due to the factthat current FDA (and other governmental agency) regulations expresslyprohibit the intentional release of recombinant antibiotic resistantbacterial strains into the environment. Hence, the utilization of theantibiotic resistant strains of bacteria disclosed in the presentinvention, produced through non-recombinant methodologies, would not beviolative of these aforementioned regulations.

[0031] Similarly, further embodiments of the present invention disclosesmethodologies for the generation of antibiotic-resistant strains oflactic acid-producing bacteria by microbial genetic- and recombinantDNA-based techniques. With respect to the microbial genetic-basedmethodology antibiotic resistance may be conferred by the “transfer” ofgenetic information from an antibiotic resistant bacterial strain to anantibiotic sensitive bacterial strain through plasmid- andnon-plasmid-mediated genetic transfer. Plasmids are small,non-chromosomal, autonomously replicating, circular DNA which oftencarry the antibiotic resistance genes. For example, in one embodiment ofthe present invention, conjugative transposons (i.e., DNA sequences thatallow the direct transfer of resistance genes without a plasmidintermediary) may be utilized to confer antibiotic resistance to anantibiotic sensitive bacterial stain. In another embodiment, recombinantDNA-based, plasmid-mediated methodologies may also be utilized.

[0032] These novel, antibiotic resistant bacterial isolates will then beused in combination with an appropriate antibiotic for the mitigation ofpathogen-associated disease and/or the reestablishment of normaldigestive flora following the administration of antibiotics and/or otheragents which deplete the gastrointestinal ecology. Hence, the presentinvention demonstrates that all antibiotic compounds possess the abilityto work synergistically with an antibiotic-resistant biorational toincrease the overall efficacy of antibiotic administration, whileconcomitantly mitigating deleterious side-effects.

[0033] In another embodiment of the present invention, the beneficial,antibiotic resistant, lactic acid-producing bacterial strain isco-administered with an anti-fungal agent and/or an antibiotic so as toameliorate the growth of both the mycotic and/or bacterial pathogen. Inaddition, antiviral agents, as well as agents which inhibit the growthof yeast may also be utilized, with or without the concomitantadministration of an antibiotic.

[0034] In yet another embodiment of the present invention, theadministration of the beneficial, lactic acid-producing bacterial strainis, by way of example but not of limitation, topical, vaginal,intra-ocular, intra-nasal, intra-otic, buccal, and the like.

[0035] 2. Use of Probiotic Bacteria to Inhibit Colonization ofAntibiotic-Resistant Gastrointestinal Pathogens

[0036] Additionally disclosed herein are compositions and methods oftreatment which exploit the novel discovery that specific, lacticacid-producing bacteria (e.g., Bacillus coagulans) possess the abilityto exhibit inhibitory activity in preventing and reducing thecolonization rates of gastrointestinal bacterial infections,particularly those infections associated with antibiotic resistantpathogens such as Enterococccus, Clostridium, Escherichia, andStaphylococcus species, as well as mitigating the deleteriousphysiological effects of the infection by the pathogen. Exceptionallyhardy or enteric-coated lactic acid-producing bacterium are preferablyused, with spore-forming Bacillus species, particularly Bacilluscoagulans, being a preferred embodiment. The present invention alsodiscloses therapeutic compositions, therapeutic systems, and methods ofuse for the treatment and/or prevention of various pathogenic bacterialgastrointestinal tract infections, particularly those infectionsassociated with antibiotic-resistant pathogens.

[0037] In one embodiment of the present invention, a therapeuticcomposition comprising a viable, non-pathogenic lactic acid-producingbacterium, preferably Bacillus coagulans, in apharmaceutically-acceptable carrier suitable for oral administration tothe gastrointestinal tract of a human or animal, is disclosed. Inanother embodiment, a Bacillus coagulans strain is included in thetherapeutic composition in the form of spores. In another embodiment, aBacillus coagulans strain is included in the composition in the form ofa dried cell mass.

[0038] In another aspect of the present invention, a composition scomprising an extracellular product of a lactic acid-producing bacterialstrain, preferably Bacillus coagulans, in a pharmaceutically-acceptablecarrier suitable for oral administration to a human or animal, isdisclosed. In a preferred embodiment, the extracellular product is asupernatant or filtrate of a culture of an isolated Bacillus coagulansstrain.

[0039] Another aspect of the invention is a method of preventing ortreating a bacterial gastrointestinal infection in a human, comprisingthe steps of orally administering to a human subject a food or drinkformulation containing viable colony forming units of a non-pathogeniclactic acid bacterium, preferably a Bacillus species and more preferablyan isolated Bacillus coagulans strain, and allowing the bacteria to growin the human subject's gastrointestinal tract.

[0040] In one embodiment of the aforementioned method, the step ofallowing the nonpathogenic bacteria to grow, further includes inhibitinggrowth of antibiotic-resistant Candida species, Staphylococcus species,Streptococcus species, Proteus species, Pseudomonas species, Escherichiacoli, Clostridium species, Klebsiella species, and Enterococccusspecies. In a preferred embodiment, the method inhibitsantibiotic-resistant Pseudomonas aeruginosa, Staphylococcus aureus,Staphylococcus pyogenes, Clostridium perfingens, Clostridium dificile,Clostridium botulinum, Clostridium tributrycum, Clostridium sporogenes,Enterococccus faecalis, Enterococccus faecium, and various othersignificant species of antibiotic gastrointestinal pathogens orcombinations thereof.

[0041] One aspect of the invention is a lactic acid-producing bacterialcomposition comprising an isolated Bacillus species strain, combinedwith a pharmaceutically-acceptable carrier suitable for oraladministration to a human or animal, wherein the isolated Bacillusspecies strain is capable of growing at temperatures of about 30° C. toabout 65° C., produces L(+) dextrorotatory lactic acid, produces sporesresistant to heat up to 90° C., and exhibits competitive, antibiotic, orparasitical activity that inhibits or reduces the colonization rate ofthe pathogenic bacteria associated with gastroenteritis and othersignificant digestive pathogens. The probiotic activity primarilyresults from vegetative growth of the isolated Bacillus species strainin the gastrointestinal tract of a human or animal. This growth causes adirect competition with the pathogenic bacteria, as well as producing anacidic, non-hospitable environment. In yet another embodiment, theprobiotic activity results from an extracellular product of the isolatedlactic acid-producing strain produced within the gastrointestinal. Thepresent invention also discloses a therapeutic system for treating,reducing or controlling gastrointestinal bacterial infections,particularly infections associated with antibiotic-resistant pathogens.

[0042] The present invention provides several advantages. In particular,insofar as there is a detrimental effect to the use of antibioticsbecause of the potential to produce antibiotic-resistant microbialspecies, it is desirable to have an anti-microbial therapy which doesnot utilize conventional anti-microbial agents. Hence, the presentinvention does not contribute to the production of future generations ofantibiotic-resistant pathogens.

[0043] 3. Use of Probiotic Bacteria in Animals

[0044] It has now been discovered that parasites and pathogenscolonizing the intestinal tract of animals can be inhibited and/orcontrolled by the use of diatomaceous earth in combination with the useof a probiotic lactic acid producing bacteria.

[0045] The present invention describes compositions, therapeuticsystems, and methods of use for inhibiting pathogen and/or parasitegrowth in the gastrointestinal tract and feces of animals. A compositionof this invention comprises an effective amount of diatomaceous earth incombination with a non-pathogenic lactic acid-producing bacteria, withspore-forming Bacillus species, particularly Bacillus coagulans, being apreferred embodiment.

[0046] According to the invention, there is provided a compositioncomprising diatomaceous earth in combination with a lacticacid-producing bacteria in a pharmaceutically- ornutritionally-acceptable carrier suitable for oral administration to thedigestive tract of an animal. In one embodiment of the composition, aBacillus coagulans strain is included in the composition in the form ofspores. In another embodiment, a Bacillus coagulans strain is includedin the composition in the form of a dried cell mass. In anotherembodiment, a Bacillus coagulans strain is included in the compositionin the form of a stabilized paste. In another embodiment, a Bacilluscoagulans strain is included in the composition in the form ofstabilized gel. In another embodiment, a Bacillus coagulans strain isincluded in the composition in the form of a stabilized liquidsuspension.

[0047] In one embodiment, the invention contemplates a compositioncomprising diatomaceous earth comprised predominantly of the Melosiragenus, preferably at least 80%. In one embodiment, the bacterial ispresent in the composition at a concentration of approximately 1×10³ to1×10¹⁴ colony forming units (CFU)/gram, preferably approximately 1×10⁵to 1×10¹² CFU/gram, whereas in other preferred embodiments theconcentrations are approximately 1×10⁹ to 1×10¹³ CFU/gram, approximately1×10⁵ to 1×10⁷ CFU/g, or approximately 1×10⁸ to 1×10⁹ CFU/gram. In oneembodiment, the bacteria is in a pharmaceutically acceptable carriersuitable for oral administration to an animal, preferably, as a powderedfood supplement, a variety of pelletized formulations, or a liquidformulation. In one embodiment, the composition further includes aneffective amount of a bifidogenic oligosaccharide, such as a short orlong chain fructo-oligosaccharide (FOS), a gluco-oligosaccharide (GOS)or other long-chain oligosaccharide polymer not readily digested bypathogenic bacteria as described herein.

[0048] The invention also describes a therapeutic system for inhibitingpathogen and/or parasite growth in the gastrointestinal tract and/orfeces of an animal comprising a container comprising a label and acomposition as described herein, wherein said label comprisesinstructions for use of the composition for inhibiting pathogen and/orparasite growth.

[0049] It should be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the present invention asclaimed.

DESCRIPTION OF THE FIGURES

[0050]FIG. 1 illustrates, in tabular form, a summary of the metabolicgrowth characteristics and requirements of Bacillus coagulans.

[0051]FIG. 2 illustrates, in tabular form, the ability of Bacilluscoagulans to inhibit various fungal pathogens, of the Trichophytonspecies, using an in vitro assay. The ATCC Accession Numbers of eachfungal strain of the Trichophyton species from the American Type CultureCollection (ATCC) are also enumerated herein.

[0052]FIG. 3 illustrates, in tabular form, the ability of Bacilluscoagulans to inhibit various yeast pathogens, of the Candida species,using an in vitro assay. The ATCC Accession Numbers of each yeast strainof the Candida species from the American Type Culture Collection (ATCC)are also enumerated herein.

[0053]FIG. 4 illustrates, in tabular form, Formulations 1-6 of thetherapeutic compositions of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0054] Unless defined otherwise, all scientific and technical terms usedherein have the same meaning as commonly understood by those skilled inthe relevant art. Unless mentioned otherwise, the techniques employed orcontemplated herein are standard methodologies well known to one ofordinary skill in the art. The examples of embodiments are forillustration only.

[0055] The present invention is discloses the recent discovery thatnon-pathogenic, lactic acid-producing bacterial species (i.e.,“probiotic bacteria”), such as the exemplary Bacillus coagulans, may beutilized in combination with antibiotic compounds or other functionalanti-microbial drugs and supplements so as to form therapeuticcompositions for use in ameliorating and/or controlling the colonizationof pathogenic bacteria with the gastrointestinal tract of both humansand animals. In addition, these non-pathogenic, lactic acid-producing,probiotic bacteria may be co-administered with an anti-fungal agentand/or an antibiotic to ameliorate the growth of the mycotic orbacterial pathogen in question. In brief, the present invention utilizesantibiotic-resistant, non-pathogenic bacteria to mitigate the growth andsubsequent establishment of antibiotic-resistant, pathogenic microbeswithin, for example, the gastrointestinal tract. Also disclosed hereinare various therapeutic compositions, methods for using said therapeuticcompositions, and systems for containing and administering/deliveringsaid therapeutic compositions.

[0056] In addition, the present invention the present invention alsodiscloses compositions and methodologies for the utilization of thesecompositions, comprising non-pathogenic, probiotic lactic acid-producingbacteria, in the mitigation of the deleterious physiological effects ofgastrointestinal tract pathogens, including antibiotic-resistantgastrointestinal tract pathogens, in both humans and animals, by thecolonization (or more-correctly, re-colonization) of thegastrointestinal tract with probiotic microorganisms, following theadministration of antibiotics, anti-fungal, anti-viral, and similaragents.

[0057] 1. Antibiotic Administration and Biorational Therapy

[0058] Antibiotics are widely used to control pathogenic microorganismsin both humans and animals. Unfortunately, the indiscriminate use ofthese agents has led to the generation of pathogenic bacteria whichfrequently exhibit resistance to multiple antibiotics. In addition, theadministration of antibiotics often results in the killing of many ofthe beneficial microorganisms (i.e., flora) within the gastrointestinaltract which contribute to “normal” gastrointestinal function (e.g.,digestion, absorption, vitamin production, and the like). Accordingly,relapse (the return of infections and their associated symptoms) andsecondary opportunistic infections often result from the depletion ofLactobacillus and other types of beneficial microbial flora within thegastrointestinal tract. Unfortunately, most, if not all, lacticacid-producing or probiotic bacteria are extremely sensitive to commonantibiotic compounds. Therefore, during a normal course of antibiotictherapy, many individuals develop a number of deleterious physiologicalside-effects including: diarrhea, intestinal cramping, and sometimesconstipation. These side-effects are primarily due to the non-selectiveaction of antibiotics, as antibiotics do not possess the ability todiscriminate between beneficial bacteria and pathogenic bacteria. Hence,both pathogenic and non-pathogenic bacteria are killed by these agents.

[0059] A biorational therapy that includes an antibiotic and anappropriate microorganism that is resistant to the selected antibioticwould serve to enhance the efficacy of the antibiotic (if the antibioticis used for the purpose of controlling a gastrointestinal tractpathogen) and assist in providing a digestive environment which isconducive to the reestablishment of the endogenous lactic acid bacteriaand suppress the growth of pathogens.

[0060] It should also be noted that the present invention is not limitedsolely to oral administration of the therapeutic compounds disclosedherein. For example, antibiotic and anti-fungal resistance is alsoassociated with topical and intra-vaginal medications. Thus, in anadditional embodiment, the co-administration of a lactic acid or otherbeneficial bacterial culture with a vaginal anti-fungal medication wouldeffectively aid in the mitigation of the mycotic or bacterial pathogenin question and repopulate the vagina and reduce the incidence ofrelapse. It should be noted that it has been demonstrated that theabsence of lactic acid-producing bacteria within the vagina is the mostcommon etiology of vaginal yeast infections and bacterial vaginosis.

[0061] In an additional embodiment, skin creams, lotions, gels and thelike could similarly contain a beneficial biorational component thatwould be effective in controlling pathogenic organisms on the skin andfurther reduce the emergence of antibiotic resistant pathogens. By wayof example, but not of limitation, the cells, spores or extracellularmaterials from such beneficial biorational bacteria could beincorporated into these skin products for this express purpose. Bumpatients usually are given antibiotics to reduce the incidence ofopportunistic infection. Pathogenic Pseudomonas, Staphylococcus, and/orEnterococci are frequently associated with infections of severe burns.Hence, the salves, lotions, gels and the like combined with thebeneficial, biorational microorganisms or their extracellular products,as disclosed in the present invention, would be effective in achieving astate of proper biodiversity to the skin in bum cases, as, generally,such biodiversity is not associated with pathogenic overgrowth.

[0062] A further embodiment of the present invention involves theutilization of probiotic organisms in livestock production, in whichantibiotics such as Vancomycin and Gentamicin are commonly used tostimulate health and weight gain. Most, if not all, probiotic organismsare sensitive to these two antibiotics and this fact has limited thepotential use of such microorganisms in the livestock industry. Inaddition, there are many environmentally-related problems associatedwith the use of antibiotics in livestock production. For example,antibiotic laden animal waste degrades very slowly and the antibioticresidue can persist, further slowing biodegradation. With the additionof species of bacteria that are resistant to Vancomycin, Gentamicin, andother antibiotics, biodegradation could actually be enhanced.

[0063] 2. Probiotic, Lactic Acid-Producing Bacterial Strains

[0064] A biorational therapy which includes an antibiotic and anappropriate microorganism that is resistant to the selected antibioticserves to both enhance the overall therapeutic efficacy of theantibiotic (if the antibiotic is used for the purpose of controlling adigestive pathogen) and to assist in providing a gastrointestinalenvironment that is conducive to the reestablishment of the endogenouslactic acid-producing bacteria and to suppress the concomitant growth ofpathogenic microorganisms.

[0065] As utilized herein, “probiotic” refers to microorganisms thatform at least a part of the transient or endogenous flora and therebyexhibit a beneficial prophylactic and/or therapeutic effect on the hostorganism. Probiotics are generally known to be clinically safe (i.e.,nonpathogenic) by those individuals skilled in the art. By way ofexample, and not of limitation to any particular mechanism, theprophylactic and/or therapeutic effect of a lactic acid-producingbacteria of the present invention results, in part, from a competitiveinhibition of the growth of pathogens due to: (i) their superiorcolonization abilities; (ii) parasitism of undesirable microorganisms;(iii) the production of lactic acid and/or other extracellular productspossessing anti-microbial activity; or (iv) various combinationsthereof. It should be noted that the aforementioned products andactivities of the lactic acid-producing bacteria of the presentinvention act synergistically to produce the beneficial probiotic effectdisclosed herein.

[0066] A probiotic bacteria which is suitable for use in the methods andcompositions of the present invention: (i) possesses the ability toproduce lactic acid; (ii) demonstrates beneficial function within thegastrointestinal tract; and is non-pathogenic. By way of example and notof limitation, many suitable bacteria have been identified and aredescribed herein, although it should be noted that the present inventionis not to be limited to currently-classified bacterial species insofaras the purposes and objectives as disclosed. The physiochemical resultsfrom the in vivo production of lactic acid is key to the effectivenessof the probiotic lactic acid-producing bacteria of the presentinvention. Lactic acid production markedly decreases the pH (i.e.,increases acidity) within the local micro-floral environment and doesnot contribute to the growth of many undesirable,physiologically-deleterious bacteria and fungi. Thus, by the mechanismof lactic acid production, the probiotic inhibits growth of competingpathogenic bacteria.

[0067] Typical lactic acid-producing bacteria useful as a probiotic ofthis invention are efficient lactic acid producers which includenon-pathogenic members of the Bacillus genus which produce bacteriocinsor other compounds which inhibit the growth of pathogenic organisms.Exemplary lactic acid-producing, non-pathogenic Bacillus speciesinclude, but are not limited to: Bacillus coagulans; Bacillus coagulansHammer; and Bacillus brevis subspecies coagulans.

[0068] Exemplary lactic acid-producing Lactobacillus species include,but are not limited to: Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasserii,Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus,bulgaricus, Lactobacillus salivarius and Lactobacillus sporogenes (alsodesignated as Bacillus coagulans).

[0069] Exemplary lactic acid-producing Sporolactobacillus speciesinclude all Sporolactobacillus species, for example, SporolactobacillusP44.

[0070] Exemplary lactic acid-producing Bifidiobacterium species include,but are not limited to: Bifidiobacterium adolescentis, Bifidiobacteriumanimalis, Bifidiobacterium bifidum, Bifidiobacterium bifidus,Bifidiobacterium breve, Bifidiobacterium infantis, Bifidiobacteriuminfantus, Bifidiobacterium longum, and any genetic variants thereof.

[0071] Several Bacillus species which are preferred in the practice ofthe present invention, include, but are not limited to the lacticacid-producing Bacillus coagulans and Bacillus laevolacticus. Variousother non-lactic acid-producing Bacillus species may be utilized in thepresent invention so long as they produce compounds which possess theability to inhibit pathogenic bacterial or mycotic growth. Examples ofsuch suitable non-lactic acid-producing Bacillus include, but are notlimited to: Bacillus subtilis, Bacillus uniflagellatus, Bacilluslateropsorus, Bacillus laterosporus BOD, Bacillus megaterium, Bacilluspolymyxa, Bacillus licheniformis, Bacillus pumilus, and Bacillussterothermophilus. Other strains that could be employed due to probioticactivity include members of the Streptococcus (Enterococcus) genus. Forexample, Enterococcus faecium, is commonly used as a livestock probioticand, thus, could be utilized as a co-administration agent. It should benoted that, although exemplary of the present invention, Bacilluscoagulans is only utilized herein as a model for various otheracid-producing (e.g., lactic acid) species of probiotic bacteria whichmay be useful in the practice of the present invention, and therefore isnot to be considered as limiting. Furthermore, it is also intended thatany of the acid-producing species of probiotic or nutritional bacteriacan be used in the compositions, therapeutic systems and methods of thepresent invention.

[0072] The Bacillus species, particularly those species having theability to form spores (e.g., Bacillus coagulans), are a preferredembodiment of the present invention. The ability to sporulate makesthese bacterial species relatively resistant to heat and otherconditions, provides for a long shelf-life in product formulations, andis deal for survival and colonization of tissues under conditions of pH,salinity, and the like within the gastrointestinal tract. Moreover,additional useful properties of many Bacillus species include beingnon-pathogenic, aerobic, facultative and heterotrophic, thus renderingthese bacterial species safe and able to readily colonize thegastrointestinal tract.

[0073] Exemplar methods and compositions are described herein usingBacillus coagulans ATCC No. 31284 (and new variants or mutants thereof)as a probiotic. Purified Bacillus coagulans is particularly useful as aprobiotic in the present invention as it is generally accepted that thevarious “classic” Lactobacillus and/or Bifidiobacterium species areunsuitable for colonization of the gut due to their instability in thehighly acidic environment of the gastrointestinal tract, particularlythe human gastrointestinal tract. In contrast, the preferred Bacillusspecies of the present invention are able to survive and colonize thegastrointestinal tract in a highly efficacious manner. Additionally,probiotic Bacillus coagulans is non-pathogenic and is generally regardedas safe (i.e., GRAS classification) by the U.S. Federal DrugAdministration (FDA) and the U.S. Department of Agriculture (USDA), andby those individuals skilled within the art.

[0074] Because Bacillus coagulans possesses the ability to produceheat-resistant spores, it is particularly useful for makingpharmaceutical compositions which require heat and pressure in theirmanufacture. Accordingly, formulations that include the utilizationviable Bacillus coagulans spores in a pharmaceutically-acceptablecarrier are particularly preferred for making and using compositionsdisclosed in the present invention.

[0075] The growth of these various Bacillus species to form cellcultures, cell pastes, and spore preparations is generally well-knownwithin the art. It should be noted that the exemplary culture andpreparative methods which are described herein for Bacillus coagulansmay be readily utilized and/or modified for growth and preparation ofthe other (lactic) acid-producing bacteria disclosed in the presentinvention.

[0076] 3. Characteristics and Sources of Bacillus coagulans

[0077] The Gram positive rods of Bacillus coagulans have a cell diameterof greater than 1.0 μm with variable swelling of the sporangium, withoutparasporal crystal production. Bacillus coagulans is a non-pathogenic,Gram positive, spore-forming bacteria that produces L(+) lactic acid(dextrorotatory) under homo-fermentation conditions. It has beenisolated from natural sources, such as heat-treated soil samplesinoculated into nutrient medium (see e.g., Bergey's Manual of SystemicBacteriology, Vol. 2, Sneath, P. H. A. et al., eds., Williams & Wilkins,Baltimore, Md., 1986). Purified Bacillus coagulans strains have servedas a source of enzymes including endonucleases (e.g., U.S. Pat. No.5,200,336); amylase (U.S. Pat. No. 4,980,180); lactase (U.S. Pat. No.4,323,651) and cyclo-malto-dextrin glucano-transferase (U.S. Pat. No.5,102,800). Bacillus coagulans has also been utilized to produce lacticacid (U.S. Pat. No. 5,079,164). A strain of Bacillus coagulans (alsoreferred to as Lactobacillus sporogenes; Sakaguti & Nakayama, ATCC No.31284) has been combined with other lactic acid producing bacteria andBacillus natto to produce a fermented food product from steamed soybeans(U.S. Pat. No. 4,110,477). Bacillus coagulans strains have also beenused as animal feeds additives for poultry and livestock to reducedisease and improve feed utilization and, therefore, to increase growthrate in the animals (International PCT Pat. Applications No. WO 9314187and No. WO 9411492). In particular, Bacillus coagulans strains have beenused as general nutritional supplements and agents to controlconstipation and diarrhea in humans and animals.

[0078] The purified Bacillus coagulans bacteria utilized in the presentinvention are available from the American Type Culture Collection (ATCC,Rockville, Md.) using the following accession numbers: Bacilluscoagulans Hammer NRS 727 (ATCC No. 11014); Bacillus coagulans Hammerstrain C (ATCC No. 11369); Bacillus coagulans Hammer (ATCC No. 31284);and Bacillus coagulans Hammer NCA 4259 (ATCC No. 15949). PurifiedBacillus coagulans bacteria are also available from the DeutscheSarumlung von Mikroorganismen und Zellkuturen GmbH (Braunschweig,Germany) using the following accession numbers: Bacillus coagulansHammer 1915 (DSM No. 2356); Bacillus coagulans Hammer 1915 (DSM No.2383, corresponds to ATCC No. 11014); Bacillus coagulans Hammer (DSM No.2384, corresponds to ATCC No. 11369); and Bacillus coagulans Hammer (DSMNo. 2385, corresponds to ATCC No. 15949). Bacillus coagulans bacteriacan also be obtained from commercial suppliers such as SabinsaCorporation (Piscataway, N.J.) or K.K. Fermentation (Kyoto, Japan).

[0079] These aforementioned Bacillus coagulans strains and their growthrequirements have been described previously (see e.g., Baker, D. et al,1960. Can. J. Microbiol. 6: 557-563; Nakamura, H. et al, 1988. Int. J.Svst. Bacteriol. 38: 63-73. In addition, various strains of Bacilluscoagulans can also be isolated from natural sources (e.g., heat-treatedsoil samples) using well-known procedures (see e.g., Bergey 's Manual ofSystemic Bacteriology, Vol. 2, p. 1117, Sneath, P. H. A. et al., eds.,Williams & Wilkins, Baltimore, Md., 1986).

[0080] It should be noted that Bacillus coagulans had previously beenmis-characterized as a Lactobacillus in view of the fact that, asoriginally described, this bacterium was labeled as Lactobacillussporogenes (See Nakamura et al. 1988. Int. J. Syst. Bacteriol. 38:63-73). However, initial classification was incorrect due to the factthat Bacillus coagulans produces spores and through metabolism excretesL(+)-lactic acid, both aspects which provide key features to itsutility. Instead, these developmental and metabolic aspects requiredthat the bacterium be classified as a lactic acid bacillus, andtherefore it was re-designated. In addition, it is not generallyappreciated that classic Lactobacillus species are unsuitable forcolonization of the gut due to their instability in the harsh (i.e.,acidic) pH environment of the bile, particularly human bile. Incontrast, Bacillus coagulans is able to survive and colonize thegastrointestinal tract in the bile environment and even grown in thislow pH range. In particular, the human bile environment is differentfrom the bile environment of animal models, and heretofore there has notbeen any accurate descriptions of Bacillus coagulans growth in humangastrointestinal tract models.

[0081] 3.1 Culture of Vegetative Bacillus Coagulans

[0082]Bacillus coagulans is aerobic and facultative, and is typicallycultured at pH 5.7 to 6.8, in a nutrient broth containing up to 2% (bywt) NaCl, although neither NaCl, nor KCl are required for growth. A pHof about 4.0 to about 7.5 is optimum for initiation of sporulation(i.e., the formation of spores). The bacteria are optimally grown at 30°C. to 45° C., and the spores can withstand pasteurization. Additionally,the bacteria exhibit facultative and heterotrophic growth by utilizing anitrate or sulfate source. The metabolic characteristics of Bacilluscoagulans are summarized in FIG. 1.

[0083]Bacillus coagulans can be cultured in a variety of media, althoughit has been demonstrated that certain growth conditions are moreefficacious at producing a culture which yields a high level ofsporulation. For example, sporulation is demonstrated to be enhanced ifthe culture medium includes 10 mg/l of MgSO₄ sulfate, yielding a ratioof spores to vegetative cells of approximately 80:20. In addition,certain culture conditions produce a bacterial spore which contains aspectrum of metabolic enzymes particularly suited for the presentinvention (i.e., production of lactic acid and enzymes for the enhancedprobiotic activity and biodegradation). Although the spores produced bythese aforementioned culture conditions are preferred, various othercompatible culture conditions which produce viable Bacillus coagulansspores may be utilized in the practice of the present invention.

[0084] Suitable media for the culture of Bacillus coagulans include: PDB(potato dextrose broth); TSB (tryptic soy broth); and NB (nutrientbroth), which are all well-known within the field and available from avariety of sources. In one embodiment of the present invention, mediasupplements which contain enzymatic digests of poultry and/or fishtissue, and containing food yeast are particularly preferred. Apreferred supplement produces a media containing at least 60% protein,and about 20% complex carbohydrates and 6% lipids. Media can be obtainedfrom a variety of commercial sources, notably DIFCO (Newark, N.J.); BBL(Cockeyesville, Md.); Advanced Microbial Systems (Shakopee, Minn.); andTroy Biologicals (Troy, Md.

[0085] In a preferred embodiment of the present invention, a culture ofBacillus coagulans Hammer bacteria (ATCC No. 31284) was inoculated andgrown to a cell density of about 1×10⁸-10⁹ cells/ml in nutrient brothcontaining: 5.0 g Peptone; 3.0 g Meat Extract; 10-30 mg MnSO₄ and 1,000ml distilled water, the broth was then adjusted to pH 7.0. The bacteriawere cultured by utilization of a standard airlift fermentation vesselat 30° C. The range of MnSO₄ acceptable for sporulation was found to be1.0 mg/l to 1.0 g/l. The vegetative bacterial cells can activelyreproduce up to 65° C., and the spores are stable up to 90° C.

[0086] Following culture, the Bacillus coagulans Hammer bacterial cellsor spores were collected using standard methods (e.g., filtration,centrifugation) and the collected cells and spores may subsequently belyophilized, spray dried, air dried or frozen. As described herein, thesupernatant from the cell culture can be collected and used as anextracellular agent secreted by Bacillus coagulans which possessesanti-microbial activity useful in a formulation of this invention.

[0087] A typical yield obtained from the aforementioned culturemethodology is in the range of approximately 1×10⁹ to 1×10¹³ viablecells/spores and, more typically, approximately 1×10¹¹ to 1.5×10¹¹cells/spores per gram prior to being dried. It should also be noted thatthe Bacillus coagulans spores, following a drying step, maintain atleast 90% viability for up to 7 years when stored at room temperature.Hence, the effective shelf-life of a composition containing Bacilluscoagulans Hammer spores at room temperature is approximately 10 years.

[0088] 3.2 Preparation of Bacillus coagulans Spores

[0089] Alternately, a culture of dried Bacillus coagulans Hammerbacteria (ATCC No. 31284) spores was prepared as follows. Approximately1×10⁷ spores were inoculated into one liter of culture mediumcontaining: 24 g (wt./vol.) potato dextrose broth; 10 g of anenzymatic-digest of poultry and fish tissue; 5 g offructo-oligosaccharides (FOS); and 10 g MnSO₄. The culture wasmaintained for 72 hours under a high oxygen environment at 37° C. so asto produce a culture having approximately 15×10¹⁰ cells/gram of culture.The culture was then filtered to remove the liquid culture medium andthe resulting bacterial pellet was resuspended in water and lyophilized.The lyophilized bacteria were ground to a fine “powder” by use ofstandard good manufacturing practice (GMP) methodologies.

[0090] It should be noted that the most preferred embodiment of thepresent invention utilizes Bacillus coagulans in spore, rather thanvegetative bacterial form.

[0091] 3.3 Preparation of B. coagulans Extracellular Products

[0092] Although the primary focus of the present invention is upon theutilization of lactic acid-producing probiotic bacteria in the form ofvegetative cells or spores, an additional embodiment utilizesextracellular products comprising a supernatant or filtrate of a cultureof a Bacillus coagulans strain for the prevention and/or control ofinfections caused by bacterium, fungi, yeast, and virus, andcombinations thereof. Extracellular products of Bacillus coagulans mayalso be included in compositions such as foods and liquids to be fed toinfants.

[0093] One liter cultures of Bacillus coagulans was prepared asdescribed in Section 5.1, except that the fructo-oligosaccharide (FOS)was omitted. The culture was maintained for 5 days as described, atwhich time FOS was added at a concentration of 5 g/liter, and theculture was continued. Subsequently, 20 ml of carrot pulp was then addedat day 7, and the culture was harvested when the culture becamesaturated (i.e., no substantial cell division).

[0094] The culture was first autoclaved for 30 minutes at 250° F., andthen centrifuged at 4000 r.p.m. for 15 mm. The resulting supernatant wascollected and filtered in a Buchner funnel through a 0.8 μm filter. Thefiltrate was collected and further filtered through a 0.2 μm Nalgevacuum filter. The resulting final filtrate was then collected (anapproximate volume of 900 ml) to form a liquid containing anextracellular product which may be further purified and/orquantitatively analyzed by use of various methodologies which arewell-known within the art.

[0095] 4 Bifidogenic Oligosaccharides

[0096] Bifidogenic oligosaccharides, as designated herein, are a classof carbohydrates particularly useful for preferentially promoting thegrowth of a lactic acid-producing bacteria of the present invention.These oligosaccharides include, but are not limited to:fructo-oligosaccharides (FOS); gluco-oligosaccharides (GOS); otherlong-chain oligosaccharide polymers of fructose and/or glucose; and thetrisaccharide-raffinose. All of these aforementioned carbohydrates arenot readily digested by pathogenic bacteria. Thus. the preferentialgrowth of lactic acid-producing bacteria is promoted by the utilizationof these bifidogenic oligosaccharides due to the nutrient requirementsof this class of bacterium, as compared to pathogenic bacteria.

[0097] Bifidogenic oligosaccharides are long chain polymers that areutilized almost exclusively by the indigenous Bifidobacteria andLactobacillus in the intestinal tract and can be similarly utilized byBacillus. In contrast, physiologically deleterious bacteria such asClostridium, Staphylococcus, Salmonella and Escherichia coli cannotmetabolize FOS, or other bifidogenic oligosaccharides, and therefor useof these bifidogenic oligosaccharides in combination with a lacticacid-producing bacteria of the present, preferably Bacillus coagulans,allows these beneficial, probiotic bacteria to grow and effectivelycompete with, and eventually replace any undesirable, pathogenicmicroorganisms within the gastrointestinal tract.

[0098] The use of bifidogenic oligosaccharides in the compositions ofthe present invention provides a synergistic effect thereby increasingthe effectiveness of the probiotic-containing compositions disclosedherein. This synergy is manifested by selectively increasing the abilityof the probiotic bacterium to grow by, for example, increasing the levelof nutrient supplementation which preferentially selects for growth ofthe probiotic bacteria over many other bacterial species within theinfected tissue.

[0099] In addition, it is readily understood that Bifidobacteria andLactobacillus are also producers of lactic acid. Bifidogenicoligosaccharides enable these aforementioned probiotic organisms toproliferate preferentially over the undesirable bacteria within thegastrointestinal tract, thereby augmenting the probiotic state of thebody by further enhancing the solubility of these nutrients (whether offood origin or as a result of nutritional supplement augmentation).Thus, the presence of the bifidogenic oligosaccharides in thecompositions of the present invention allows for more effectivemicrobial inhibition by increasing the ability of all varieties ofprobiotic bacteria to grow, and therefore provide said benefit.

[0100] The bifidogenic oligosaccharide of the present invention may beused either alone, or in combination with a lactic acid-producingmicroorganisms in a therapeutic composition. More specifically, due tothe growth promoting activity of bifidogenic oligosaccharides, thepresent invention contemplates a composition comprising a bifidogenicoligosaccharide present in a concentration sufficient to promote thegrowth of lactic acid-producing microorganisms. As shown herein, theseconcentrations amounts can vary widely, as the probiotic microorganismswill respond to any metabolic amount of nutrient oligosaccharide, andtherefore the present invention need not be so limited.

[0101] A preferred and exemplary bifidogenic oligosaccharide is FOS,although other carbohydrates may also be utilized, either alone or incombination. FOS can be obtained from a variety of natural sources,including commercial suppliers. As a product isolated from naturalsources, the components can vary widely and still provide the beneficialagent, namely FOS. FOS typically has a polymer chain length of fromabout 4 to 200 sugar units, with the longer lengths being preferred. Forexample, the degree of purity can vary widely so long asbiologically-functional FOS is present in the final formulation.Preferred FOS formulations contain at least 50% by weight offructo-oligosaccharides compared to simple(mono or disaccharide) sugarssuch as glucose, fructose or sucrose, preferably at least 80%fructo-oligosaccharides (FOS), more preferably at least 90% and mostpreferably at least 95% FOS. Sugar content and composition can bedetermined by any of a variety of complex carbohydrate analyticaldetection methods as is well known. Preferred sources of FOS include,but are not limited to: inulin; Frutafit IQ™ (Imperial Suiker Unie;Sugar Land, Tex.); NutraFlora™ (Americal Ingredients, Inc.; Anaheim,Calif.); and Fruittrimfat Replacers and Sweeteners (Emeryville, Calif.).Bifidogenic oligosaccharides such as GOS, and other long chainoligosaccharides are also available from commercial vendors.

[0102] 5. Diatomaceous Earth

[0103] Diatomaceous earth is the skeletal remains of single cell aquaticplants known as diatoms which are typically relatively uniform incomposition, depending upon the source of the deposit and the componentspecies of diatoms present in the deposit. Diatomaceous earth ischaracterized as having a silica content, a characteristic morphologicalshape, depending upon the species, and an average size of from about 5to 20 microns (μm) in diameter.

[0104] Different species of diatoms in diatomaceous earth provide adiverse range of shapes, providing different degrees of sharp and/orspiny edges which when contacted with insects, parasites and smallmicroorganisms pierce the protective coatings of the targetparasite/pathogen. Diatomaceous earth is included in a therapeuticcomposition of this invention in a wide variety of concentrations,depending upon the manner of administration. Typical compositionscontain from about 0.1 to 99% weight of diatomaceous earth per weight(w/w) of composition. For concentrated single dose uses, a high contentof diatomaceous earth is used, typically 5 to 50% w/w, and preferablyabout 5 to 10% w/w. For continuous feed applications, a moderate to lowcontent of diatomaceous earth is used, typically 0.5 to 10% w/w, andpreferably 1 to 5% w/w.

[0105] A preferred diatomaceous earth for use in a composition of thepresent invention has a low ash content, typically less than 1% w/w, alow amorphous silica content, typically less than 1% w/w, and a lowvolconoclastic sediment, typically less than 1% w/w. Insofar as apreferred diatomaceous earth has the further property of presentingsharp and/or spiny edges to damage the external protective surfaces ofthe parasite/pathogen to be inhibited. Diatom shapes are wellcharacterized in art, and the spiny, sharp character can be easilyobserved by microscopic examination of the diatoms. By observation andquantitative analysis, one can readily determine the proportions of thecomponent diatoms in the diatomaceous earth. A preferred diatomaceousearth contains a high content of abrasive diatoms. A particularlypreferred diatomaceous earth contains Melosira diatoms, and preferablyis comprised of at least 50% w/w Melosira diatoms, more preferably atleast 70% w/w Melosira diatoms, and most preferably at least 80%Melosira diatoms.

[0106] Diatomaceous earth can be obtained from a variety of sources.Typically, any diatom deposit is a source of diatomaceous earth.Commercial suppliers routinely mine, characterize and provide differentgrades of diatomaceous earth. A particularly preferred supplier ofdiatomaceous earth rich in Melosira diatoms is the CR MineralsCorporation, Golden, Colo.

[0107] 6. Methods of Producing or Enhancing Antibiotic Resistance

[0108] As previously discussed, the present invention disclosesmethodologies for the selection, isolation, and culturing ofantibiotic-resistant strains of lactic acid-producing bacteria be usedas concomitantly administered biorational agents. These embodiments maybe predicated upon: (i) selectively culturing the probiotic bacteria(which may initially be sensitive to the antibiotic of choice) ingradually increasing concentrations of antibiotic of choice in order tofacilitate the development of decreased antibiotic sensitivity or,preferably, antibiotic resistance; (ii) utilizing “conjugativetransposons” (i.e., DNA sequences that allow the direct transfer ofresistance genes without a plasmid intermediary) to confer antibioticresistance to an antibiotic sensitive bacterial stain; and (iii)utilizing plasmids (i.e., small, non-chromosomal, autonomouslyreplicating, circular DNA which often naturally possess antibioticresistance genes) possessing genes conferring resistance to theantibiotic of choice which are generated by standard RecombinantDNA-based techniques.

[0109] It should be noted, however, that the most preferred embodimentof the present invention utilizes the selective culturing of theprobiotic bacteria in gradually increasing concentrations of antibioticof choice in order to facilitate the development of decreased antibioticsensitivity or, preferably, antibiotic resistance. This embodiment,which will be more fully discussed below, is preferred due to the factthat current FDA and other governmental agency regulations expresslyprohibit the intentional release of “man-made” (e.g., recombinant)antibiotic resistant bacterial strains into the environment. Hence, theutilization of the antibiotic resistant strains of bacteria disclosed inthe present invention, produced through the non-recombinant-based,selective culture-based methodology, would not be violative of theseaforementioned regulations. It should be noted, however, that thepreference of this embodiment is not intended to be limiting, but ratherreflects current regulations governing this field of endeavor. Shouldthese regulations be modified, or if new regulations are promulgated,the inventors fully intend to utilize all methodologies disclosed hereinto practice the present invention in the most efficacious mannerpossible.

[0110]Bacillus coagulans (strain ATCC Accession No. 31284) was assayedfor antibiotic resistance/sensitivity utilizing the Kirby-Bauer agardilution method (see e.g., Bergey's Manual of Systemic Bacteriology,Vol. 2, Sneath, P. H. A. et al., eds., Williams & Wilkins, Baltimore,Md., 1986). This methodology demonstrated that this Bacillus coagulansstrain was susceptible to Piperacillin, Trimethoprim-Sulfamethoxasole,Ampicillin, Ciprofloxacin, Erythromycin, Vancomycin, Gentamicin, andOxacillin and was intermediate with respect to Clindamycin andTetracycline. Specifically, by Vitek, the MICs were found to be: (i)Ampicillin—2; (ii) Penicillin G—0.12; (iii) Vancomycin—<0.5; (iv)Nitrofurantoin—<32; (v) Norfioxacin—<4; (vi) Chloramphenicol—8; (vii)Clindamycin—>8 (resistant); and Tetracycline—>16 (resistant). Bacilluscoagulans (strain ATCC Accession No. 31284) possesses natural resistanceto the antibiotics Clindamycin and Tetracycline.

[0111] Subsequently, each prospective microorganism was then screened,utilizing the aforementioned methodology, for antibiotic sensitivity.Media and agars which were specific for each prospective bacteria weremixed with sub-lethal levels of the desired antibiotic compound. Forexample, DIFCO Trypticase Soy Agar (TSA) containing a sub-lethal levelof Vancomycin was prepared. The media/antibiotic mixture was thensterilized by steam autoclaving, ethylene oxide, or ionizing radiation(i.e., Gamma Processing) in cases where the antibiotic in question wassensitive to extreme heat. Petri dishes containing the agar/antibioticmixture were poured and the prospective microorganisms (selected from asingle colonies) were streaked on these plates.

[0112] Surviving (i.e., viable) bacterial colonies were then selectedand transferred to new antibiotic-containing media in which theconcentration of the selected antibiotic was gradually increased totherapeutic levels. At each stage of the selection process, survivingcolonies of Bacillus coagulans were selected and transferred to newmedia until therapeutic level antibiotic resistance is established.

[0113] 7. Probiotic Activity of Bacillus coagulans

[0114] It is well-documented clinically that many species of bacterial,mycotic and yeast pathogens possess the ability to cause a variety ofgastrointestinal disorders including, but not limited to: disruption ofnormal gastrointestinal biochemical function, necrosis ofgastrointestinal tissues, and disruption of the bioabsorption ofnutrients, and like conditions. Therefore, the utilization of theprobiotic microorganism-containing compositions of the present inventioninhibits these pathogens are useful in the prophylactic or therapeutictreatment of conditions associated with infection by theseaforementioned pathogens.

[0115] 7.1 Anti-microbial Probiotic Activity

[0116] The ability of Bacillus coagulans to inhibit various bacterialpathogens was quantitatively ascertained by use of an in vitro assay.This assay is part of a standardized bacterial pathogen screen(developed by the U.S. Food and Drug Administration(FDA)) and iscommercially available on solid support disks (DIFCO® BACTROL®Antibiotic Disks). To perform the assay, potato-dextrose plates (DIFCO®)were initially prepared using standard procedures. The plates were thenindividually inoculated with the bacteria (approximately 1.5×10⁶ CFU) tobe tested so as to form a confluent bacterial bed.

[0117] Inhibition by Bacillus coagulans was subsequently ascertained byplacing approximately 1.8×10⁶ CFU of Bacillus coagulans in 10 μl ofbroth or buffer, directly in the center of the potato-dextrose platewith one test locus being approximately 8 mm in diameter per plate. Aminimum of three test loci were used for each assay. The negativecontrol consisted of a 10 μl volume of a sterile saline solution,whereas the positive control consisted of a 1 μl volume ofglutaraldehyde. The plates were then incubated for approximately about18 hr at 30° C., at which time the zones of inhibition were measured. Asdesignated herein, “excellent inhibition” means the zone was 10 mm orgreater in diameter; and “good inhibition” means the zone was greaterthan 2 mm in diameter but less than 10 mm in diameter.

[0118] As expected, no “inhibition” was seen with the negative, salinecontrol, and excellent “inhibition” (approximately 16.2 mm diameter;average of three tests) was seen with the positive, glutaraldehydecontrol. For the enteric microorganisms tested, the following inhibitionby Bacillus coagulans was found: (i) Clostridium species—excellentinhibition; (ii) Escherichia coli—excellent inhibition; (iii)Clostridium species—excellent inhibition, where the zone of inhibitionwas consistently greater than 15 mm in diameter. Similarly, excellentinhibition was also seen for the opportunistic pathogens Pseudornonasaeruginosa, and Staphylococcus aureus. In summation, pathogenic entericbacteria which were shown to be inhibited by Bacillus coagulans activityinclude, but are not limited to: Staphylococcus aureus; Staphylococcusepidermidis; Streptococcus pyogenes; Pseudomonas aeruginosa; Escherichiacoli (enterohemorragic species); numerous Clostridium species (e.g.,Clostridium perfingens, Clostridium botulinum, Clostridium tributrycum,Clostridium sporogenes, and the like); Gardnereia vaginails;Proponbacterium aenes; Aeromonas hydrophia; Aspergillus species; Proteusspecies; and Klebsiella species.

[0119] 7.2 Anti-Mycotic Probiotic Activity

[0120] The ability of Bacillus coagulans to inhibit various fungalpathogens was demonstrated using an in vitro assay. The tested fungalstrains of Trichophyton species are available from the American TypeCulture Collection (ATCC; Rockville, Md.) and their ATCC accessionnumbers are illustrated in FIG. 2.

[0121] In the assay, potato-dextrose plates (DIFCO®, Detroit, Mich.)were prepared using standard procedures and were inoculated individuallywith a confluent bed (about 1.7×10⁶) of various species of the fungusTrichophyton. Inhibition by Bacillus coagulans was ascertained byplacing on the plate approximately 1.5×10⁶ colony forming units (CFU) in10 μl of broth or buffer, plated directly in the center of thepotato-dextrose plate, with one test locus per plate. The size of eachtest locus was approximately 8 mm in diameter and a minimum of threetests were performed for each inhibition assay. The negative controlconsisted of a 10 ml volume of sterile saline solution, whereas thepositive control consisted of a 10 ml volume 2% Miconazole(1-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxylmethyl-1,11-imidazole within an inert cream.

[0122] The plates were then incubated for approximately 18 hr at 30° C.,at which time the zones of inhibition were measured. As designatedherein, “excellent inhibition” means the zone was 10 mm or greater indiameter; and “good inhibition” means the zone was greater than 2 mm indiameter, but less than 10 mm in diameter.

[0123] The results of in vitro inhibition by Bacillus coagulans areillustrated in FIG. 2. For each of the Trichophyton species tested, thedisease condition associated with an infection is indicated in column 2of FIG. 2. For comparison, no zone of inhibition was seen with thenegative control, whereas good inhibition (approximately 8.5 mmdiameter, mean average of three tests) was seen with the positivecontrol.

[0124] 7.3 Probiotic Inhibition of Yeast

[0125] Similarly, the ability of Bacillus coagulans to inhibit variousyeast pathogens was demonstrated in vitro for four species of Candida,all of which are available from the American Type Culture Collection(ATCC; Rockville, Md.) with their ATCC accession numbers illustrated inFIG. 3.

[0126] In the assay, potato-dextrose plates (DIFCO®, Detroit, Mich.)were prepared using standard procedures and were inoculated individuallywith a confluent bed about 1.7×10⁶ of the four species of Candida.Inhibition by B. coagulans was tested by placing on the plate about1.5×10⁶ colony forming units (CFU) in 10 μl of broth or buffer, plateddirectly in the center of the potato-dextrose plate with one test locusof about 8 mm in diameter per plate. A minimum of three tests wereperformed for each inhibition assay. The negative control consisted of a1 ml volume of a sterile saline solution, whereas the positive controlconsisted of a 1 ml volume of Miconazole cream.

[0127] The plates were then incubated for approximately 18 hr at 30° C.,at which time the zones of inhibition were measured. As designatedherein, “excellent inhibition” means the zone was 10 mm or greater indiameter; and “good inhibition” means the zone was greater than 2 mm indiameter, but less than 10 mm in diameter.

[0128] The results of the in vitro tests are shown in FIG. 3 with thepathological conditions in humans associated with infection by theCandida species shown in column 2. As expected, no inhibition was seenwith the negative control and good inhibition (approximately 8.7 mmdiameter; average of three tests) was seen with the positive control.

[0129] 8. Therapeutic Compositions

[0130] 8.1 Anti-Microbial Agent-Containing Therapeutic Compounds

[0131] It should be noted that although Bacillus coagulans is utilizedherein as a preferred exemplary probiotic species, by virtue of thecommon physiological characteristics which are indigenous to all lacticacid-producing bacteria, other species of these lactic acid-producingbacteria may be effectively in the methods and/or therapeuticcompositions disclosed in the present invention. Preferred, exemplaryformulations of the therapeutic compositions of the present inventionare set forth in FIG. 4.

[0132] The cells/spores can be presented in a variety of compositionssuited for oral administration to the gastrointestinal tract, directedat the objective of introducing the bacteria to tissues of thegastrointestinal tract. Therapeutic compositions of the presentinvention are, for example, comprised of a lactic acid-producingbacteria strain, preferably vegetative Bacillus coagulans, Bacilluscoagulans spores, or combinations thereof which are a co-administratedwith a selected agents which possesses the ability to ameliorateinfections which have a bacterial, fungal, and/or yeast etiology. In theaforementioned embodiment, the active lactic acid-producing bacteriaspecies of the present invention comprise approximately 0. 1% to 50% byweight of the final composition and, preferably, approximately 1% to 10%by weight, contained within a formulation suitable for oraladministration. More specifically, the therapeutic composition of thepresent invention may contain, within a 350 mg dosage formulation, forexample, approximately 2×10⁶ to 1×10¹⁰ colony forming units (CFU) ofviable, lactic acid-producing vegetative bacteria or bacterial spores(in the case of Bacillus coagulans).

[0133] The formulation for a therapeutic composition of the presentinvention may also include other probiotic agents or nutrients whichpromote spore germination and/or bacterial growth. A particularlypreferred material is a bifidogenic oligosaccharide, which promotes thegrowth of beneficial probiotic bacteria as previously described, supra.Bifidogenic oligosaccharides (e.g., fructo-oligosaccharide (FOS) orgluco-oligosaccharide (GOS)) may be utilized in various combinations,depending upon the specific formulation. The preferred therapeuticcomposition includes approximately 10 to 200 mg of bifidogenicoligosaccharide, and most preferably a concentration of approximately100 to 500 mg of bifidogenic oligosaccharide per unit of the therapeuticcomposition. Additionally, the therapeutic composition of the presentinvention may include other probiotic agents or nutrients for promotinggrowth, as well as other physiologically-active constituents which donot interfere with the overall therapeutic efficacy of the other activeagents contained within the therapeutic composition.

[0134] In another embodiment of the present invention, the Bacilluscoagulans strain is combined with a therapeutically-effective dose of an(preferably, broad-spectrum) antibiotic. The therapeutic composition ofthe present invention may also contain approximately 1 to approximately250 mg of the selected antibiotic per unit of therapeutic composition.In preferred embodiments of the present invention, the Bacilluscoagulans strain is combined with a therapeutic dose of an antibioticsuch as Gentamicin; Vancomycin; Oxacillin; Tetracyclines;Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-Sulfamethoxasole; a member of the Cephlosporin antibioticfamily (e.g., Cefaclor, Cefadroxil, Cefixime, Cefprozil, Ceftriaxone,Cefuroxime, Cephalexin, Loracarbef, and the like); a member of thePenicillin family of antibiotics (e.g., Ampicillin,Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin, Penicillin VK, andthe like); with a member of the Fluoroquinolone family of antibiotics(e.g., Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); or amember of the Macrolide antibiotic family (e.g., Azithromycin,Erythromycin, and the like).

[0135] In another embodiment of the present invention, the Bacilluscoagulans strain is combined with a therapeutically-effective dose of ananti-fungal agent. The therapeutic composition of the present inventionmay also contain approximately 1 to 250 mg of the selected anti-fungalagent per unit of therapeutic composition. Typical anti-fungal agentswhich may be utilized include, but are not limited to: Clotrimazole,Fluconazole, Itraconazole, Ketoconazole, Miconazole, Nystatin,Terbinafine, Terconazole, Tioconazole, and the like.

[0136] In a preferred embodiment, Bacillus coagulans spores, atherapeutically-effective concentration of an antibiotic, anti-fungal,etc., and, if so desired, various other components (e.g., bifidogenicoligosaccharide, binders, etc.) are encapsulated into anenterically-coated, time-released capsule or tablet. The enteric coatingallows the capsule/tablet to remain intact (i.e., undisolved) as itpasses through the gastrointestinal tract, until such time as it reachesthe small intestine. Similarly, the time-released component prevents the“release” of the Bacillus coagulans spores for a pre-determined timeperiod which, preferably, will coincide with the end of the antibiotictreatment period as the antibiotic prevents the spores from geminatinguntil such time as the serum levels drop to a substantially low level.Once the antibiotic regimen is completed, the Bacillus coagulans sporesgerminate and this microorganism becomes the primary resident flora ofthe gastrointestinal tract, due to the killing-off of the previousresident flora by the antibiotic.

[0137] In addition, the vegetative Bacillus coagulans microorganisms donot adhere to the intestinal epithelium. Thus (without a repeat dosage),the bacteria remain in the gastrointestinal tract for maximal time ofapproximately 10 days and are considered to be a transient flora. Therelatively rapid gastrointestinal-clearance time and inability to adhereto the gastrointestinal epithelium of Bacillus coagulans, has theadvantage of preventing the later development of bacteremia in (forexample) immunocompromised individuals.

[0138] The therapeutic compositions of the present invention may alsoinclude known antioxidants, buffering agents, and other agents such ascoloring agents, flavorings, vitamins or minerals. For example, apreferred therapeutic composition may also contain one or more of thefollowing minerals: calcium citrate (15-350 mg); potassium gluconate(5-150 mg); magnesium citrate (5-15 mg); and chromium picollinate (5-200μg). In addition, a variety of salts may be utilized, including calciumcitrate, potassium gluconate, magnesium citrate and chromiumpicollinate. Thickening agents may be added to the compositions such aspolyvinylpyrrolidone, polyethylene glycol or carboxymethylcellulose.Preferred additional components of a therapeutic composition of thisinvention can include assorted colorings or flavorings, vitamins, fiber,enzymes and other nutrients. Preferred sources of fiber include any of avariety of sources of fiber including, but not limited to: psyllium,rice bran, oat bran, corn bran, wheat bran, fruit fiber and the like.Dietary or supplementary enzymes such as lactase, amylase, glucanase,catalase, and the like enzymes can also be included. Chemicals used inthe present compositions can be obtained from a variety of commercialsources, including Spectrum Quality Products, Inc (Gardena, Calif.),Sigma Chemicals (St. Louis, Mich.), Seltzer Chemicals, Inc., (Carlsbad,Calif.) and Jarchem Industries, Inc., (Newark, N.J.).

[0139] The various active agents (e.g., probiotic bacteria, antibiotics,anti-fungal agents, bifidogenic oligosaccharides, and the like) arecombined with a carrier which is physiologically compatible with thegastrointestinal tissue of the species to which it is administered.Carriers can be comprised of solid-based, dry materials for formulationinto tablet, capsule or powdered form; or the carrier can be comprisedof liquid or gel-based materials for formulations into liquid or gelforms. The specific type of carrier, as well as the final formulationdepends, in part, upon the selected route(s) of administration.

[0140] The therapeutic composition of the present invention may alsoinclude a variety of carriers and/or binders. A preferred carrier ismicro-crystalline cellulose (MCC) added in an amount sufficient tocomplete the one gram dosage total weight. Particularly preferredformulations for a therapeutic composition of this invention will bedescribed, infra. Carriers can be solid-based dry materials forformulations in tablet, capsule or powdered form, and can be liquid orgel-based materials for formulations in liquid or gel forms, which formsdepend, in part, upon the routes of administration.

[0141] Typical carriers for dry formulations include, but are notlimited to: trehalose, malto-dextrin, rice flour, micro-crystallinecellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose,sucrose, and like carriers. Where the composition is dry and includesevaporated oils that produce a tendency for the composition to cake(adherence of the component spores, salts, powders and oils), it ispreferred to include dry fillers which distribute the components andprevent caking. Exemplary anti-caking agents include MCC, talc,diatomaceous earth, amorphous silica and the like, and are typicallyadded in an amount of from approximately 1% to 95% by weight. It shouldalso be noted that dry formulations which are subsequently rehydrated(e.g., liquid formula) or given in the dry state (e.g., chewable wafers,pellets or tablets) are preferred to initially hydrated formulations.Dry formulations (e.g., powders) may be added to supplement commerciallyavailable foods (e.g., liquid formulas, strained foods, or drinkingwater supplies). Similarly, the specific type of formulation dependsupon the route of administration.

[0142] Suitable liquid or gel-based carriers include but are not limitedto: water and physiological salt solutions; urea; alcohols andderivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g.,ethylene glycol, propylene glycol, and the like). Preferably,water-based carriers possess a neutral pH value (i.e., pH 7.0). Thecompositions may also include natural or synthetic flavorings andfood-quality coloring agents, all of which must be compatible withmaintaining viability of the lactic acid-producing microorganism.Well-known thickening agents may also be added to the compositions suchas corn starch, guar gum, xanthan gum, and the like. Where aliquid-based composition containing spores is provided, it is desirableto include a spore germination inhibitor to promote long term storage.Any spore germination inhibitor may be used. By way of example and notof limitation, preferred inhibitors include: hyper-saline carriers,methylparaben, guargum, polysorbates, preservatives, and the like.

[0143] Preservatives may also be included within the carrier includingmethylparaben, propylparaben, benzyl alcohol and ethylene diaminetetraacetate salts. Well-known flavorings and/or colorants may also beincluded within the carrier. The compositions of the present inventionmay also include a plasticizer such as glycerol or polyethylene glycol(with a preferred molecular weight of MW=800 to 20,000). The compositionof the carrier can be varied so long as it does not interferesignificantly with the pharmacological activity of the activeingredients or the viability of the Bacillus coagulans spores.

[0144] A composition can be formulated to be suitable for oraladministration in a variety of ways, for example in a liquid, a powderedfood supplement, a paste, a gel, a solid food, a packaged food, a wafer,and the like. Other formulations will be readily apparent to one skilledin the art.

[0145] A nutrient supplement component of a composition of thisinvention can include any of a variety of nutritional agents, as arewell known, including vitamins, minerals, essential and nonessentialamino acids, carbohydrates, lipids, foodstuffs, dietary supplements, andthe like. Preferred compositions comprise vitamins and/or minerals inany combination. Vitamins for use in a composition of this invention caninclude vitamins B, C, D, E, folic acid, K, niacin, and like vitamins.The composition can contain any or a variety of vitamins as may bedeemed useful for a particularly application, and therefore, the vitamincontent is not to be construed as limiting. Typical vitamins are those,for example, recommended for daily consumption and in the recommendeddaily amount (RDA), although precise amounts can vary. The compositionwould preferably include a complex of the RDA vitamins, minerals andtrace minerals as well as those nutrients that have no established RDA,but have a beneficial role in healthy human or mammal physiology. Thepreferred mineral format would include those that are in either thegluconate or citrate form because these forms are more readilymetabolized by lactic acid bacteria. In a related embodiment, theinvention contemplates a composition comprising a viable lactic acidbacteria in combination with any material to be adsorbed, including butnot limited to nutrient supplements, foodstuffs, vitamins, minerals,medicines, therapeutic compositions, antibiotics, hormones, steroids,and the like compounds where it is desirable to insure efficient andhealthy absorption of materials from the gastrointestinal track into theblood. The amount of material included in the composition can varywidely depending upon the material and the intended purpose for itsabsorption, such that the invention is not to be considered as limiting.Other components of the compositions of the present invention can be abifidogenic oligosaccharide, as described herein.

[0146] By way of example, and not of limitation, Bacillus coagulansspores may be incorporated into any type of dry or lyophilized productwhich is dissolved or mixed with hot water, so long as the temperatureof the Bacillus coagulans spore-containing mixture is raised to therequired heat-shock temperature (i.e., 80° C. for 5 minutes) necessaryfor germination of the spores. The Bacillus coagulans spores may eitherbe incorporated into the dry or lyophilized product by the manufacturerof the product or by the consumer during preparation. These dry orlyophilized product include, but are not limited to: tea bags, coffee(e.g., “freeze-dried” or ground), sweeteners (e.g., synthetic(NutraSweet®) and natural); hot cereal (e.g., oatmeal, Cream of Wheat®,and the like), hot beverage condiments/flavorings and creamers, and thelike.

[0147] In another specific embodiment, Bacillus coagulans spores may beutilized as a dry or lyophilized product, or incorporated into achewable tablet, toothpaste, mouthwash, oral drops, and the like inorder to inhibit the formation of dental caries, gingivitis, and otherforms of periodontal disease. Similarly, Bacillus coagulans spores maybe incorporated, with or without anti-microbial agents, chewable tablet,toothpaste, mouthwash, oral drops, and the like in order to treat oralinfections caused by yeast (i.e., “thrush”), Herpes simplex I (i.e.,cold sores), and various other infections caused by oral pathogens.

[0148] In yet another specific embodiment, the Bacillus coagulansvegetative bacterial cells/spores may incorporated into an aqueoussolution (e.g., physiological saline) for administration as a colonic,via an enema or the like) so as to directly administer the probioticbacteria to the colon. This method of administration is highlyefficacious for utilization of vegetative bacterial cells as they arenot exposed to the highly acidic environment of the stomach as is thecase during oral administration.

[0149] 8.2 Therapeutic Compositions Methods for Treating BacterialInfections

[0150] The present invention contemplates a method for treating,reducing or controlling gastrointestinal bacterial infections using thetherapeutic composition or therapeutic system disclosed herein. Thedisclosed methods of treatment function so as to inhibit the growth ofthe pathogenic bacteria which are associated with gastrointestinalinfections, as well as to concomitantly mitigate the deleteriousphysiological effects/symptoms of these pathogenic infections.

[0151] Probiotic lactic acid bacterium, preferably Bacillus coagulans,are generally regarded as safe by those skilled within the art (i.e.,GRAS Certified by the FDA) and, therefore, suitable for direct ingestionin food stuffs or as a food supplement. The methods of the presentinvention comprise administration of a therapeutic compositioncontaining a viable lactic acid-producing bacteria to thegastrointestinal tract of a human or animal, to treat or preventbacterial infection. Administration is preferably made using a liquid,powder, solid food and the like formulation compatible with oraladministration, all formulated to contain a therapeutic composition ofthe present invention by use of methods well-known within the art.

[0152] The methods of the present invention includes administration of acomposition containing lactic acid-producing bacterial cells (i.e.,vegetative bacterial cells) and/or spores or isolated Bacillus coagulansextracellular products (which contains a metabolite possessingantibiotic-like properties) to a human or animal, so as to treat orprevent the colonization of antibiotic-resistant pathogens with thegastrointestinal tract. In particular, for VRE, VISA, PRP, and otherpathogens, the methods includes administering to the patient, forexample, Bacillus coagulans in food or as a food supplement. Oraladministration is preferably in an aqueous suspension, emulsion, powderor solid, either already formulated into a food, or as a compositionwhich is added to food by the user prior to consumption. Administrationto the gastrointestinal tract may also be in the form of an analsuppository (e.g., in a gel or semi-solid formulation). All suchformulations are made using standard methodologies.

[0153] Administration of a therapeutic composition is preferably to thegastrointestinal tract using a gel, suspension, aerosol spray, capsule,tablet, powder or semi-solid formulation (e.g., a suppository)containing a therapeutic composition of the present invention, allformulated using methods well-known within the art. Administration ofthe compositions containing the active probiotic lactic acid-producingbacterium which is effective in preventing or treating a pathogenicbacterial infection, generally consist of one to ten dosages ofapproximately 10 mg to 10 g of the therapeutic composition per dosage,for a time period ranging from one day to one month. Administrations are(generally) once every twelve hours and up to once every four hours. Inthe preferred embodiment, two to four administrations of the therapeuticcomposition per day, of approximately 0.1 g to 5 g per dose, for one toseven days. This preferred dose is sufficient to prevent or treat apathogenic bacterial infection. Of course, the specific route, dosageand timing of the administration will depend, in part, upon theparticular pathogen and/or condition being treated, as well as theextent of said condition.

[0154] A preferred embodiment of the present invention involves theadministration of from approximately 1×10³ to 1×10¹⁴ CFU of viable,vegetative bacteria or spore per day, more preferably from approximately1×10⁵ to 1×10¹⁰, and most preferably from approximately 5×10⁸ to 1×10⁹CFU of viable, vegetative bacteria or spores per day. Where thecondition to be treated involves antibiotic-resistant digestivepathogens and the patient is an adult, the typical dosage isapproximately 1×10² to 1×10¹⁴ CFU of viable, vegetative bacteria orspores per day, preferably from approximately 1×10⁸ to 1×10¹⁰, and morepreferably from approximately 2.5×10⁸ to 1×10¹⁰ CFU of viable,vegetative bacteria or spores per day. Where the condition to be treatedis Sudden Infant Death Syndrome (SIDS) and the patient is an infant over6 months old, the dosage is typically 1×10⁶ to 1×10⁹, preferably fromapproximately 5×10⁴ to 2.5×10⁵, and more preferably from approximately1.5×10⁵ to 2×10⁵ CFU of viable, vegetative bacteria or spores per day.

[0155] In addition, the present invention contemplates a method whichcomprises oral administration of a composition that contains fromapproximately 10 mg to 20 g of a bifidogenic oligosaccharide, preferablya fructo-oligosaccharide (FOS), per day, preferably from approximately50 mg to 10 g, and more preferably from approximately 150 mg to 5 g perday, to preferentially promote the growth of the probiotic lacticacid-producing bacterium over the growth of the pathogen. The method canbe combined with treatment methods using a probiotic lacticacid-producing bacterium as described herein.

[0156] The present invention further contemplates a therapeutic systemfor treating, reducing and/or controlling pathogenic bacterialinfections. Typically, the system is in the form of a package containinga therapeutic composition of the present invention, or in combinationwith packaging material. The packaging material includes a label orinstructions for use of the components of the package. The instructionsindicate the contemplated use of the packaged component as describedherein for the methods or compositions of the invention.

[0157] By way of example, and not of limitation, a system can compriseone or more unit dosages of a therapeutic composition according to thepresent invention. Alternatively, the system can alternately containbulk quantities of a therapeutic composition. The label containsinstructions for using the therapeutic composition in either unit doseor in bulk forms as appropriate, and may also include informationregarding storage of the composition, disease indications, dosages,routes and modes of administration and the like information.

[0158] Furthermore, depending upon the particular contemplated use, thesystem may optionally contain either combined or in separate packagesone or more of the following components: bifidogenic oligosaccharides,flavorings, carriers, and the like components. One particularlypreferred embodiment comprises unit dose packages of Bacillus spores foruse in combination with a conventional liquid product, together withinstructions for combining the probiotic with the formula for use in atherapeutic method.

[0159] 9. Utilization of the Therapeutic Compositions of the PresentInvention in the Treatment of Bacterial Gastroenteritis

[0160] Several microbial species have been quantitatively ascertained asthe etiology for the vast majority of food-borne gastrointestinalinfection (i.e., bacterial gastroenteritis), with Campylobacterjejuni-mediated campylobacteriosis being the most commonly reported(46%) cause of bacterial gastroenteritis in the United States, followedin prevalence by Salmonella typhimurium-mediated salmonellosis (28%);shigellosis (17%); and Escherichia coli O157 infection (5%). Inaddition, it is quite possible that various Salmonella and Shigellaspecies may eventually acquire antibiotic resistance (i.e., Metacillinor Vancomycin) in that same manner in which Enterococci originallyacquired antibiotic resistance from Staphylococcus aureus.

[0161] Although the methodologies disclosed in the present invention areequally applicable to the therapeutic intervention of all forms ofbacterial gastroenteritis, by way of example and not of limitation, thefollowing discussion will be primarily limited to the utilization ofthese methodologies in the treatment of Campylobacter jejuni-mediatedbacterial gastroenteritis.

[0162]Campylobacter jejuni was first identified as a humangastrointestinal tract (i.e., diarrheal) pathogen in 1973. As previouslystated, in 1996, 46% of all laboratory-confirmed cases of bacterialgastroenteritis reported to the Centers for Disease Control andPrevention (CDC) were caused by Campylobacter species. In the UnitedStates alone, an estimated 2.1 to 2.4 million cases of humancampylobacteriosis occur each year. See e.g., Tauxe, R. V. Epidemiologyof Campylobacter jejuni infections in the United States and otherindustrial nations. In: Campylobacter jejuni: current and future trends.P. 9-13 (Nachamkin, I. and Tompkins L. S., editors; American Society forMicrobiology; 1992). Less frequently, Campylobacter jejuni infectionshave also been reported to cause bacteremia, septic arthritis, andvarious other extra-intestinal pathology. See e.g., Peterson, M. C.,1994. Wes. J. Med. 161: 148-152. In addition, an increasing proportionof human infections caused by Campylobacter jejuni are resistant toanti-microbial therapy. The mishandling of raw poultry and consumptionof undercooked poultry are the major risk factors for humancampylobacteriosis.

[0163] Deaths from Campylobacter jejuni-related infections arerelatively rare, and occur primarily in infants, the elderly, andindividuals with underlying illnesses. For example, the incidence ofcampylobacteriosis in HIV-positive/AIDS patients is markedly higher thanin the general population. In Los Angeles County between 1983 and 1987,the reported incidence of campylobacteriosis in patients with AIDS was519 cases per 100,000 population, which is 39-times higher than the ratein the general population. See e.g., Sorvillo, F. J. et al., 1991. J.Acquired Immune Defic. Syndr. Hum. Retrovirol. 4: 595-602. Commoncomplications of campylobacteriosis in HIV-infected individuals includerecurrent infections with antimicrobial-resistant bacterial strains. Seee.g., Penman, D. J. et al., 1988. Ann. Intern. Med. 108: 540-546.

[0164] 9.1 Pathophysiology of Campylobacter jejuni-MediatedGastroenteritis

[0165] The pathophysiology of Campylobacter jejuni-mediatedgastroenteritis involves both host- and pathogen-specific factors.Factors including, but not limited to, the overall health and age of thehost (see e.g., Tauxe, R. V. Epidemiology of Campylobacter jejuniinfections in the United States and other industrial nations. In:Campylobacter jejuni: current and future trends. P. 9-13 (Nachamkin, I.and Tompkins L. S., editors; American Society for Microbiology; 1992)and Campylobacter jejuni-specific humoral immunity from previousexposure (see e.g., Blaser, M. J. et al., 1987. JAMA 257: 43-46)influence the clinical outcome following infection.

[0166] The ingestion of relatively low numbers of viable organisms issufficient to cause infection in healthy adults. For example, in onevolunteer study, Campylobacter jejuni infection was demonstrated tooccur after the ingestion of as few as 800 organisms, with the overallrates of infection increasing as a function of the ingested dose. Seee.g., Black, R. E. et al., 1988. J. Infect. Dis. 157: 472-479. Inaddition, the rates of infection appeared to increase when bacterialinocula were ingested in a suspension buffered to reduce gastricacidity. See e.g., Black, R. E. et al., 1988. J. Infect. Dis. 157:472-479. Similarly, both rates of Campylobacter jejuni infectivity andthe severity of accompanying disease appeared to be positively effectedby disturbances in the overall gastrointestinal “health” of the infectedindividual (e.g., secondary disease or infection precipitating loweredlevels of normal gastrointestinal flora, and the like. In accord, thesensitivity of Campylobacter jejuni to decreased pH (i.e., acidicenvironments) and competing bacterial species serves to illustrate thepotential efficacy of the utilization of the antibiotic resistant,lactic acid-producing probiotic bacteria (in combination with theappropriate antibiotic) disclosed in the present invention to mitigatethe in vivo growth of Campylobacter jejuni, and hence its rate ofinfectivity, by generating an inhospitable acidic, competitiveenvironment within the individual's gastrointestinal tract.

[0167] Many pathogen-specific virulence determinants may contribute tothe pathogenesis of Campylobacter jejuni-mediated infection, but nonehas a quantitatively ascertained role. See e.g., Ketley, J. M., 1997.Microbiology 143: 5-21. Suspected determinants of pathogenicity include,but are not limited to: chemotaxis, motility, and flagella, all of whichare required for attachment and colonization of the gastrointestinalepithelium. See e.g., Ketley, J. M., 1997. Microbiology 143: 5-21. Oncecolonization occurs, other possible virulence determinants are ironacquisition, host cell invasion, toxin production, inflammation andactive secretion, and epithelial disruption with leakage of serosalfluid. See e.g., Ketley, J. M., 1997. Microbiology 143: 5-21.

[0168] 9.2 Sequelae to Campylobacter jejuni Infection

[0169] Several chronic, extra-gastrointestinal sequelae are associatedwith Campylobacter jejuni-mediated bacterial gastroenteritis (e.g.,Guillain-Barré syndrome and Reiter syndrome—a reactive type ofarthropathy).

[0170] Guillain-Barré syndrome (GBS), a demyelating disorder resultingin acute neuromuscular paralysis, is a serious sequelae of Campylobacterinfection. See e.g., Allos, B. M., 1997. J. Infect. Dis. 176: 5125-5128.It has been estimated that one case of GBS occurs for every 1,000 casesof campylobacteriosis and up to 40% of patients with the syndrome havedemonstrated evidence of recent Campylobacter infection. Approximately20% of patients with GBS are left with some disability, andapproximately 5% die despite recent advances in respiratory care.

[0171] Campylobacteriosis is also associated with Reiter syndrome, areactive arthropathy. See e.g., Peterson, M. C., 1994. Scand. J.Rheumatol. 23: 167-170. In approximately 1% of patients withcampylobacteriosis, the sterile post-infection process occurs 7 to 10days after onset of diarrhea. Multiple joints can be affected,particularly the knee joint. Pain and incapacitation can last for monthsor, in some cases, become chronic. Both GBS and Reiter syndrome arethought to be autoimmune responses stimulated by infection. For example,many individuals with Reiter syndrome have been found to carry the HLAB27 antigenic marker. See e.g., Peterson, M. C., 1994. Scand. J.Rheumatol. 23: 167-170. Unfortunately, the pathogenesis of GBS (seee.g., Shoenfeld, Y. et al., 1996. Int. Arch. Allergy Immunol. 109:318-326) and Reiter syndrome is not completely understood.

[0172] 9.3 Anti-Microbial-Resistant Strains of Campylobacter jejuni

[0173] The increasing rate of human infections caused byanti-microbial-resistant strains of Campylobacter jejuni makes clinicalmanagement of cases of campylobacteriosis markedly more difficult. Seee.g., Murphy, G. S. et al., 1996. Clin. Infect. Dis. 22: 568-569;Piddock, L. V., 1995. Antimicrob. Agents Chemother. 36: 891-898. Aspreviously discussed, anti-microbial resistance can prolong illness andcompromise treatment of patients with bacteremia. Interestingly, therate of anti-microbial-resistant Campylobacter jejuni-mediated entericinfections is highest in the developing world, where the use ofanti-microbial drugs in humans and animals is relatively unrestricted.For example, a 1994 study found that most clinical isolates ofCampylobacter jejuni from United States troops in Thailand weredemonstrated to be resistant to the broad spectrum antibiotic,Ciprofloxacin. Similarly, approximately one-third of Campylobacterjejuni isolates from United States troops located in Hat Yai were alsofound to be resistant to Azithromycin. See e.g., Murphy, G. S. et al.,1996. Clin. Infect. Dis. 22: 568-569. In the industrialized world, theemergence of Campylobacter jejuni strains which have been found to beresistant to the broad spectrum antibiotic Fluoroquinolone, isillustrative of the need for prudent anti-microbial use in food-animalproduction. See e.g., Piddock, L. V., 1995. Antimicrob. AgentsChemother. 36: 891-898. Experimental evidence demonstrates thatFluoroquinolone-susceptible Campylobacter jejuni readily becomedrug-resistant in poultry when these drugs are administered. See e.g.,Jacobs-Reitsma, W. F. et al., The induction of quinolone resistance inCampylobacter bacteria in broilers by quinolone treatment. In:Campylobacter, Helicobacters, and related organisms. 1996. (Newell, D.G., Ketley, J. M., and Feldman, R. A., editors. New York: Plenum Press)p. 307-11. After Fluoroquinolone use in poultry was approved in Europe,resistant Campylobacter jejuni strains were shown to rapidly emerge inhumans during the early 1990's. See e.g., Piddock, L. V., 1995.Antimicrob. Agents Chemother. 36: 891-898. Similarly, within 2 years ofthe 1995 approval of Fluoroquinolone use for poultry in the UnitedStates, the number of domestically-acquired human cases ofCiprofloxacin-resistant campylobacteriosis doubled in Minnesota. In a1997 study conducted in Minnesota, (20%) of 60 Campylobacter jejuniisolates obtained from chicken purchased in grocery stores were found tobe Ciprofloxacin-resistant. See e.g., Smith, K. E. et al.,Fluoroquinolone-resistant Campylobacter isolated from humans and poultryin Minnesota. 1995. Program of the 1st International Conference onEmerging Infectious Diseases; Mar. 7-10, 1998. Centers for DiseaseControl and Prevention; Atlanta, Ga.

[0174] 9.4 Treatment of Campylobacter jejuni-Mediated Infections

[0175] Current, traditional therapeutic modalities primarily involvesupportive measures, particularly fluid and electrolyte replacement, formost patients with campylobacteriosis. See e.g., Blaser, M. J.,Campylobacter Species. In: Principles and practice of infectiousdiseases. 1990. p. 1649-1658 (Mandell, G. L., ed., ChurchhillLivingstone). Severely dehydrated patients should receive rapid volumeexpansion with intravenous fluids, however for most other patients, oralrehydration is indicated.

[0176] Although Campylobacter infections are generally self-limiting innature, antibiotic therapy may be prudent for patients who have highfever, bloody diarrhea, or more than eight stools in 24 hours;immunosuppressed patients, patients with systemic infections, and thosewhose symptoms worsen or persist for more than 1 week from the time ofinitial diagnosis. When indicated, anti-microbial therapy soon after theonset of symptoms can reduce the median duration of illness fromapproximately 10 days to 5 days. However, when such treatment is delayed(e.g., until Campylobacter jejuni infection is confirmed by a medicallaboratory), antibiotic therapy may not be successful. Ease ofadministration, lack of serious toxicity, and high degree of efficacymake erythromycin the drug of choice for Campylobacter jejuni infection;however, other anti-microbial agents, particularly the quinolones andnewer Macrolides (e.g., Azithromycin) may also utilized.

[0177] The utilization of antibiotic agents, which kill the “normal”microbial flora, frequently exacerbates the deleterious physiologicaleffects (e.g., diarrhea, loss of the gastrointestinal mucosa,dehydration, and the like) in individuals with Campylobacterjejuni-mediated bacterial gastroenteritis. Accordingly, the concomitantadministration of an antibiotic and an antibiotic resistant probioticmicroorganisms of the present invention to these individuals mayameliorate these aforementioned deleterious physiological symptomologyby re-establishing the gastrointestinal microbial flora which serves toboth directly compete with the pathogenic bacteria for required growthmoieties (e.g., lipids, carbohydrates, electrolytes, amino acids, andthe like), as well as making the gastrointestinal environmentinhospitable to the continued growth of the pathogenic bacteria bylowering the pH through the production of lactic acid.

[0178] 9.5 Other Bacterial Gastrointestinal Pathogens

[0179] Various other gastrointestinal pathogens, some antibioticresistant, have been recently reported. These pathogens are amenable forprevention or treatment with the present invention.

[0180] For example, the FDA is investigating whether bacteria resistantto quinolone antibiotics can emerge in food animals and cause disease inhumans. Although thorough cooking has been demonstrated to sharplyreduce the likelihood of antibiotic-resistant bacteria surviving in meatinfect a human, pathogens resistant to drugs other than fluoroquinoloneshave been sporadically reported to survive in meat and subsequentlyinfect a human. In 1983, for example, 18 people in four midwesternstates developed multi-drug-resistant Salmonella food poisoning aftereating beef from cows fed antibiotics. Eleven of the people werehospitalized, and one died.

[0181] A study conducted by Cometta, et al., showed that increase inantibiotic resistance parallels increase in antibiotic use in humans.See e.g., Cometta, et al., 1994. New Engl. J. Med. 126: 43-47. Theyexamined a large group of cancer patients given fluoroquinoloneantibiotics. The patients' white blood cell counts were very low as aresult of their cancer treatment, thus leaving them open toopportunistic infection. Between 1983 and 1993, the percentage of suchpatients receiving antibiotics rose from 1.4 to 45. During those years,the researchers isolated Escherichia coli bacteria annually from thepatients, and tested the microbes for resistance to five types offluoroquinolones. Between 1983 and 1990, all 92 E. coli strains testedwere easily killed by the antibiotics. But from 1991 to 1993, 11 of 40tested strains (28 percent) were found to be resistant to all fivedrugs.

[0182] 10. Therapeutic Methods for Inhibiting Parasites in Animals

[0183] The present invention is also directed at methods for inhibitinggrowth of parasites and/or pathogenic organisms in the gastrointestinaltract of animals. The method comprises administering a composition ofthe present invention to the gastrointestinal tract of the animal, andthereby contact any parasites therein with an effective amount of theactive ingredients in the composition.

[0184] As used herein, the terms “pathogen” and “parasite” are usedinterchangeably in the context of a deleterious organism growing in thegastrointestinal tract and/or feces of an animal, although itappreciated that these terms have distinctive meanings.

[0185] The present invention describes methods for inhibiting growth ofa parasite in the gastrointestinal tract of an animal comprising thestep of administering a composition of the invention to thegastrointestinal tract of the animal. A composition preferably containsdiatomaceous earth and viable lactic acid-producing bacteria. Themetabolic effect of diatomaceous earth present in a composition of thisinvention on parasites is to rupture tissues of the parasite, typicallythe soft cuticle portions of the ectoskeleton, based on the abrasivequality of the diatomaceous earth upon cuticles arising during themechanical effects of movement of the parasite after contacting the CE.These ruptures in the cuticle breach the protective ectoskeleton of theparasite, rendering the parasite susceptible to infection, todehydration, to fluid exchanges and/or fluid losses, and the likeeffects which inhibit parasite health, and thereby inhibit growth.

[0186] The combined use of diatomaceous earth with an lacticacid-producing bacteria provides an beneficial synergy which providesimportant benefits to the claimed compositions, methods and systems. Asdescribed herein, the use of the probiotic bacterial promotes healthygrowth in the intestinal tract, competing out deleterious bacteria,making the tissues targeted by the deleterious bacteria more healthy.Parasites cause local tissue damage at the site of growth and feeding,and often provide inflammation and tissue injuries at the site as well.This tissue damage provides a pathogenic or unhealthy environment wherethe tissue is ruptured and/or compromised in health, allowingundesirable or opportunistic pathogens to grow in the tissue vicinity.Because the parasite damages tissue and creates an environment thatfavors pathogenic infections, diatomaceous earth inhibits both theparasite and the pathogenic infection by reducing the degree of tissuedamage. Because the health of the host contributes to the ability tofight off the parasite, improvements in tissue health by decreasingpathogenic infections with probiotics increase the ability to inhibitparasite growth. Thus, the probiotic and the diatomaceous earthcooperate at inhibiting pathogens and parasites, respectively, whichgrowth in turn promotes growth of each other, decreasing tissue damageand increasing digestive health of the host.

[0187] In one embodiment the invention contemplates methods forinhibiting growth of parasites and pathogenic organisms in the feces ofanimals. The method comprises administering a composition of the presentinvention into the gastrointestinal tract of an animal, therebyintroducing the active ingredients of the composition into theintestinal tract of the animal. A composition containing diatomaceousearth in an effective amount controls and/or inhibits parasite orpathogen growth in feces by first interfering with viable growth in theintestines, where the parasite first grows, thereby reducing the amountof parasite arriving in (i.e., “inoculating”) the feces, andsubsequently by interfering with growth that occurs in the feces afterthe feces is deposited.

[0188] Insofar as feces provide growth and breeding grounds forundesirable organisms, controlling and/or inhibiting growth of parasitesand pathogenic organisms in feces inhibits growth and reproduction ofthese undesirable organisms in areas where feces is produced, depositedand/or stored. For example, in barns or corrals, in animal cages, infeed lots, in zoological display enclosures, and the like areas whereanimals are maintained and feces is deposited, there is an opportunityfor parasites/pathogens to irritate, spread, reproduce and/or infectother hosts. These circumstances provide a variety of undesirableproblems solved by the present invention. For example, it is undesirablefor parasites or pathogens to spread and further infect hosts, andthereof or any means to control spread of infection is of great benefitwhere multiple animals are caged together. In addition, in manycircumstances biting of host animals by parasites or flying insectsirritates and/or upsets animals, providing behavior problems whichincludes excessive kicking, biting and related activities which areunsafe for neighboring animals and for animal handlers.

[0189] In a particularly preferred embodiment, the inventioncontemplates a method for reducing and/or controlling flying insectpopulations in animal cages/pens/enclosures where animals are maintainedcomprising administering a composition of the present invention to thegastrointestinal tract of the caged animals.

[0190] The present invention is useful at controlling a large variety ofparasites and pathogenic organisms, and therefore the invention need notbe limited to inhibiting any particular genus or species of organism.For example, based on the mechanisms described herein for effectivenessof the composition, it is seen that all insect varieties which can actas an animal parasite can be targeted by the methods of the presentinvention. Parasites can infect any of a variety of animals, includingmammals, reptiles, birds and the like, and therefore the invention isdeemed to not be limited to any particular animal. Examples ofwell-known or important parasites are described herein for illustrationof the invention, but are not to be viewed as limiting the invention.Representative parasites and animal and/or human hosts are described inextensive detail in a variety of veterinary treatises such as “Merck'sVeterinary Manual” and “Cecils' Human Diseases” Parasites of horsesincludes horse bots, lip bots or throat bots, caused by Gasterophilusspecies, such as G. intestinalis, G. haemorrhiodalis, and G. nasalis,stomach worms, caused by Habronema species, such as H. muscae or H.microstoma mulus, or caused by Crascia species, such as C. mepastoma, orcaused by Trichostrongvlus species, such as T. axei, ascarids (whiteworms) caused by Parascaris species such as P. eciuorum, blood worms(palisade worms, red worms or sclerostomes) caused by Stroncrvlusspecies such as S. vulcraris, S. epuinus or S. edentatus, smallstrongyles of the cecum and colon caused by Triodontophorus species suchas T. tenuicollis, pinworms caused by Oxvuris species such as O. eaui,strongyloides infections of the intestine caused by Stroncivloideswesteri, tapeworms caused by Anonlocephala species such as A. macma andA. perfoliata, and caused by Paranonlocephala mamillana.

[0191] Various other parasites cause disease in ruminants, typicallycattle, include the wire worm (or barber's pole worm or large stomachworm) caused by Haemonchus species. Parasites caused in ruminants,typically swine, include stomach worms caused by Hvostroncmulus species.

[0192] Additional parasites are known to infect a variety of animalhosts, and therefore are a target for treatment by the methods of thepresent invention. For example, gastrointestinal parasites infect avariety of animals and can include Spirocerca species such as S. lupithat cause esopheageal worms in canines and Physoloptera species thatcause stomach worms in canines and felines.

[0193] In humans, a large variety of parasites are particularlyimportant targets for the methods of the present invention insofar asthese parasites are well known. However, the invention is not to beconstrued as limited to these parasites.

[0194] Where the animal is fed a pelletized or granular food, thecomposition can be included in the pelletized or granular food, or cancomprise a mixture of the pelletized food combined with a pelletizedcomposition of this invention. Mixing pelletized food with a pelletizedformulation of a composition of this invention is a particularlypreferred method for practicing the present invention, insofar as itprovides a convenient system for using commercial feeds andsimultaneously regulating the amounts of a composition of this inventionto be administered.

[0195] Administration of a therapeutic composition is preferably to thegut using a gel, suspension, aerosol spray, capsule, tablet, granule,pellet, wafer, powder or semi-solid formulation (e.g., a suppository)containing a nutritional composition of this invention, all formulatedusing methods well known in the art.

[0196] The method comprises administration of a composition of thisinvention containing the active ingredients to a human or animal invarious dosage regimens as described herein to achieve the nutritionalresult. Administration of the compositions containing the activeingredients effective in inhibiting parasite growth in the intestine andin feces generally consist of one to ten unit dosages of 10 mg to 10 gper dosage of the composition for one day up to one month for an animalof approximately 100 kg body weight. Unit dosages are generally givenonce every twelve hours and up to once every four hours. Preferably twoto four dosages of the composition per day, each comprising about 0.1 gto 50 g per dosage, for one to seven days are sufficient to achieve thedesired result.

[0197] A preferred method involves the administration into the digestivetract of from 1×10² to 1×10¹⁰ viable bacterium or spore per day, in someembodiments from 1×10³ to 1×10⁶, in other embodiments from 1×10⁶ to1×10⁹, and more preferably about from 5×10⁸ to 1×10⁹ viable bacterium orspore per day. Exemplary dosages range from about 1×10³ to 1×10⁶ viablebacterium per day, or alternatively range from about 1×10⁶ to 1×10⁹viable bacterium per day.

[0198] In a related embodiment, a preferred method comprisesadministration of the composition which delivers from about 0.1 to 25%weight of diatomaceous earth per volume (w/v) of composition, where thecomposition is typically formulated as an animal feed, preferably about0.5 to 10% (w/v), and more preferably about 1 to 5% (w/v). Typically,when used in animal feed a single dose route of administration will usea higher diatomaceous earth concentration, such as about 2 to 10% w/v,preferably about 5% w/v. When an animal feed route of administration isused in a daily feed mode, a lower diatomaceous earth concentration istypically used, for example about 0.5 to 2% w/v, preferably about 1%w/v. Stated differently, a typical unit dosage is a compositioncontaining about 50 milligrams (mg) to 10 grams of diatomaceous earth,preferably from about 200 to 500 mg, per 100 kilogram animal per day.

[0199] In addition, a preferred method comprises administering into thedigestive tract from 10 mg to 20 grams of fructo-oligosaccharide perday, preferably about 50 mg to 10 grams, and more preferably about from150 mg to 5 grams of fructo-oligosaccharide per day. These dosages areexpressed for an animal of approximately 70-100 kilogram body weight.For animals of other body sizes, the dosages are adjusted according tothe above body weight to dosage ratios.

[0200] The method is typically practiced on any animal where inhibitingpathogen or parasites is desired. The animal can be any livestock orzoological specimen where such inhibition of parasites/pathogensprovides economic and health benefits. Any animal can benefit by theclaimed methods, including birds, reptiles, mammals such as horses,cows, sheep, goats, pigs, and the like domesticated animals, or any of avariety of animals of zoological interest. Other purposes are readilyapparent to one skilled in the arts of nutrient absorption, feedutilization and bioavailability.

[0201] 10.1. Therapeutic Systems for Inhibiting Parasite Growth

[0202] The present invention further contemplates a system forinhibiting growth of parasites and/or pathogens in the gastrointestinaltract of an animal or in animal feces comprising a container comprisinglabel and a composition according to the present invention, wherein saidlabel comprises instructions for use of the composition for inhibitingpathogen/parasite growth.

[0203] Typically, the system is present in the form of a packagecontaining a composition of this invention, or in combination withpackaging material. The packaging material includes a label orinstructions for use of the components of the package. The instructionsindicate the contemplated use of the package component as describedherein for the methods or compositions of the invention.

[0204] For example, a system can comprise one or more unit dosages of atherapeutic composition according to the invention. Alternatively, thesystem can contain bulk quantities of a composition. The label containsinstructions for using the composition in either unit dose or in bulkforms as appropriate, and may include information regarding storage ofthe composition, feeding instruction, health and diet indications,dosages, routes of administration, methods for blending the compositionwith pre-selected food stuffs, and the like information.

[0205] Equivalents

[0206] From the foregoing detailed description of the specificembodiments of the present invention, it should be readily apparent thata unique methodology for the utilization of lactic acid-producingbacteria, preferably Bacillus coagulans, for the prevention andtreatment of gastrointestinal tract pathogens and their associateddiseases, has been described. Although particular embodiments have beendisclosed herein in detail, this has been done by way of example forpurposes of illustration only, and is not intended to be limiting withrespect to the scope of the appended claims which follow. In particular,it is contemplated by the inventor that various substitutions,alterations, and modifications may be made to the invention withoutdeparting from the spirit and scope of the invention as defined by theclaims. For instance, the choice of the particular antibiotic which isutilized in the Therapeutic Composition of the present invention isbelieved to be a matter of routine for a person of ordinary skill in theart with knowledge of the embodiments described herein.

What is claimed is:
 1. A therapeutic composition for the treatment orprevention of a gastrointestinal infection caused by a pathogenicbacteria, comprising a therapeutically-effective concentration of one ormore species or strains of lactic acid-producing bacteria within apharmaceutically-acceptable carrier suitable for administration to thegastrointestinal tract of a vertebrate, wherein said lacticacid-producing bacteria possesses the ability to reduce both thecolonization rate and the potential physiologically deleterious effectsdue to the colonization of said pathogenic bacteria.
 2. The therapeuticcomposition of claim 1, wherein one of said lactic acid-producingbacteria is a Bacillus species which is selected from a groupcomprising: Bacillus coagulans; Bacillus coagulans Hammer; and Bacillusbrevis subspecies coagulans, and any genetic variants thereof.
 3. Thetherapeutic composition of claim 1, wherein one of said lacticacid-producing bacteria is a Lactobacillus species which is selectedfrom a group comprising: Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasserii,Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus,bulgaricus, Lactobacillus salivarius and Lactobacillus sporogenes (alsodesignated as Bacillus coagulans), and any genetic variants thereof. 4.The therapeutic composition of claim 1, wherein one of said lacticacid-producing bacteria is a Sporolactobacillus species which isselected from a group comprising: Sporolactobacillus P44, and anygenetic variants thereof.
 5. The therapeutic composition of claim 1,wherein one of said lactic acid-producing bacteria is a Bifidiobacteriumspecies which is selected from a group comprising: Bifidiobacteriumadolescentis, Bifidiobacterium animalis, Bifidiobacterium bifidum,Bifidiobacterium bifidus, Bifidiobacterium breve, Bifidiobacteriuminfantis, Bifidiobacterium infantis, Bifidiobacterium longum, and anygenetic variants thereof.
 6. A therapeutic composition for the treatmentor prevention of a gastrointestinal infection caused by a pathogenicbacteria, comprising a therapeutically-effective concentration of ananti-microbial agent selected from the group comprising an antibiotic,an anti-fungal agent, and anti-yeast agent, or an anti-viral agent, andof one or more species or strains of lactic acid-producing bacteriawithin a pharmaceutically-acceptable carrier suitable for administrationto the gastrointestinal tract of a vertebrate, wherein said lacticacid-producing bacteria possesses the ability to reduce both thecolonization rate and the potential physiologically deleterious effectsdue to the colonization of said pathogenic bacteria.
 7. The therapeuticcomposition of claim 6, wherein one of said lactic acid-producingbacteria is a Bacillus species which is selected from a groupcomprising: Bacillus coagulans; Bacillus coagulans Hammer; and Bacillusbrevis subspecies coagulans, and any genetic variants thereof.
 8. Thetherapeutic composition of claim 6, wherein one of said lacticacid-producing bacteria is a Lactobacillus species which is selectedfrom a group comprising: Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasserii,Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus,bulgaricus, Lactobacillus salivarius and Lactobacillus sporogenes (alsodesignated as Bacillus coagulans), and any genetic variants thereof. 9.The therapeutic composition of claim 6, wherein one of said lacticacid-producing bacteria is a Sporolactobacillus species which isselected from a group comprising: Sporolactobacillus P44, and anygenetic variants thereof.
 10. The therapeutic composition of claim 6,wherein one of said lactic acid-producing bacteria is a Bifidiobacteriumspecies which is selected from a group comprising: Bifidiobacteriumadolescentis, Bifidiobacterium animalis, Bifidiobacterium bifidum,Bifidiobacterium bifidus, Bifidiobacterium breve, Bifidiobacteriuminfantis, Bifidiobacterium infantis, Bifidiobacterium longum, and anygenetic variants thereof.
 11. A therapeutic composition for thetreatment or prevention of a gastrointestinal infection caused by apathogenic bacteria, comprising a therapeutically-effectiveconcentration of an anti-microbial agent, and one or more species orstrains of Bacillus coagulans within a pharmaceutically-acceptablecarrier suitable for administration to the gastrointestinal tract of avertebrate, wherein said Bacillus coagulans bacterial strains possessantibiotic resistance or decreased antibiotic sensitivity to theantibiotic selected for use in said composition and wherein saidBacillus coagulans possesses the ability to reduce both the colonizationrate and the potential physiologically deleterious effects due to thecolonization of said pathogenic bacteria.
 12. The therapeuticcomposition of claim 11, wherein said Bacillus coagulans bacterial stainis included in said composition in a form selected from a groupconsisting of a dried bacterial cell mass within a flowable concentrate,a stabilized gel, or a stabilized paste.
 13. The therapeutic compositionof claim 11, wherein said Bacillus coagulans bacterial stain is in formof a dried bacterial spore mass which possess the ability to germinatefollowing administration within a flowable concentrate, a stabilizedgel, or a stabilized paste.
 14. The therapeutic composition of claim 11,wherein the total administered concentration of said therapeuticcomposition ranges from approximately 10 milligrams to approximately 10grams of composition per day, and preferably ranges from approximately0.1 grams to approximately 5 grams of composition per day.
 15. Thetherapeutic composition of claim 11, wherein the total administeredconcentration of Bacillus coagulans within said therapeutic compositionranges from approximately 1×10³ to approximately 1×10¹⁴ viable bacteriaor spores per day, preferably ranges from approximately 1×10⁵ toapproximately 1×10¹⁰ viable bacteria or spores per day, and morepreferably ranges from approximately 5×10⁸ to approximately 1×10⁹ viablebacteria or spores per day.
 16. The therapeutic composition of claim 11,wherein the infection to be treated is within an adult and is caused byan antibiotic-resistant pathogen, and wherein the total administeredconcentration of Bacillus coagulans within said therapeutic compositionranges from approximately 1×10² to approximately 1×10¹⁴ viable bacteriaor spores per day, preferably ranges from approximately 1×10⁸ toapproximately 1×10¹⁰ viable bacteria or spores per day, and morepreferably ranges from approximately 2.5×10⁸ to approximately 1×10¹⁰viable bacteria or spores per day.
 17. The therapeutic composition ofclaim 11, wherein the infection to be treated is Sudden Infant DeathSyndrome (SIDS) in an infant over six months of age and is caused by anantibiotic-resistant pathogen, and wherein the total administeredconcentration of Bacillus coagulans within said therapeutic compositionranges from approximately 1 x 10 ⁶ to approximately 1×10⁹ viablebacteria or spores per day, preferably ranges from approximately 5×10⁴to approximately 2.5×10⁵ viable bacteria or spores per day, and morepreferably ranges from approximately 1.5×10⁵ to approximately 2×10⁵viable bacteria or spores per day.
 18. The therapeutic composition ofclaim 11, wherein said therapeutic composition additionally contains oneor more bifidogenic factors, wherein said bifidogenic oligosaccharide isselected from the group consisting of fructo-oligosaccharide (FOS),gluco-oligosaccharide (GOS), raffinose, and long-chain oligosaccharides.19. The therapeutic composition of claim 11, wherein said therapeuticcomposition additionally contains one or more bifidogenic factors andwherein said bifidogenic factor is a fructo-oligosaccharide (FOS), andwherein the total administered concentration of the bifidogenic factorwithin said therapeutic composition ranges ranging from approximately 10milligrams to approximately 20 grams per gram of therapeutic compositionper day, preferably from approximately 50 milligrams to approximately 10grams per gram of therapeutic composition per day, and more preferablyfrom approximately from approximately 150 milligrams to approximately 5grams per gram of therapeutic composition per day.
 20. The therapeuticcomposition of claim 11, wherein said pathogenic bacteria areantibiotic-resistant pathogenic bacteria.
 21. The therapeuticcomposition of claim 11, wherein the physiological location of theadministration of said therapeutic composition is selected from a groupcomprising: buccal; topical; vaginal; nasal; ocular; and oticadministration locations.
 22. The therapeutic composition of claim 11,wherein said anti-microbial agent is selected from the group comprising:antibiotics, anti-fungal agents, anti-viral agents, and anti-yeastagents.
 23. The therapeutic composition of claim 11, wherein saidanti-microbial agent is comprised of a therapeutically-effectiveconcentration of an antibiotic selected from a group consisting of:Gentamicin; Vancomycin; Oxacillin; Tetracyclines; Nitroflurantoin;Chloramphenicol; Clindamycin; Trimethoprim-Sulfamethoxasole; a member ofthe Cephlosporin antibiotic family; a member of the Penicillinantibiotic family; a member of the Fluoroquinolone antibiotic family;and a member of the Macrolide antibiotic family.
 24. The therapeuticcomposition of claim 11, wherein said anti-microbial agent is comprisedof a therapeutically-effective concentration of an anti-fungal agentselected from a group consisting of: Clotrimazole, Fluconazole,Itraconazole, Ketoconazole, Miconazole, Nystatin, Terbinafine,Terconazole, and Tioconazole.
 25. A therapeutic composition for thetreatment or prevention of a gastrointestinal infection caused by apathogenic bacteria, comprising a therapeutically-effective of one ormore species or strains of Bacillus coagulans within apharmaceutically-acceptable carrier suitable for administration to thegastrointestinal tract of a vertebrate, wherein said Bacillus coagulansbacterial strains possess the ability to reduce both the colonizationrate and the potential physiologically deleterious effects due to thecolonization of said pathogenic bacteria.
 26. The therapeuticcomposition of claim 25, wherein said Bacillus coagulans bacterial stainis included in said composition in a form selected from a groupconsisting of a dried bacterial cell mass within a flowable concentrate,a stabilized gel, or a stabilized paste.
 27. The therapeutic compositionof claim 25, wherein said Bacillus coagulans bacterial stain is in formof a dried bacterial spore mass which possess the ability to germinatefollowing administration within a flowable concentrate, a stabilizedgel, or a stabilized paste.
 28. The therapeutic composition of claim 25,wherein the total administered concentration of said therapeuticcomposition ranges from approximately 10 milligrams to approximately 10grams of composition per day, and preferably ranges from approximately0.1 grams to approximately 5 grams of composition per day.
 29. Thetherapeutic composition of claim 25, wherein the total administeredconcentration of Bacillus coagulans within said therapeutic compositionranges from approximately 1×10³ to approximately 1×10¹² viable bacteriaor spores per day, preferably ranges from approximately 1×10⁵ toapproximately 1×10¹⁰ viable bacteria or spores per day, and morepreferably ranges from approximately 5×10⁸ to approximately 1×10⁹ viablebacteria or spores per day.
 30. The therapeutic composition of claim 25,wherein the infection to be treated is within an adult and is caused byan antibiotic-resistant pathogen, and wherein the total administeredconcentration of Bacillus coagulans within said therapeutic compositionranges from approximately 1×10² to approximately 1×10¹⁴ viable bacteriaor spores per day, preferably ranges from approximately 1×10⁸ toapproximately 1×10¹⁰ viable bacteria or spores per day, and morepreferably ranges from approximately 2.5×10⁸ to approximately 1×10¹⁰viable bacteria or spores per day.
 31. The therapeutic composition ofclaim 25, wherein the infection to be treated is Sudden Infant DeathSyndrome (SIDS) in an infant over six months of age and is caused by anantibiotic-resistant pathogen, and wherein the total administeredconcentration of Bacillus coagulans within said therapeutic compositionranges from approximately 1×10⁶ to approximately 1×10⁹ viable bacteriaor spores per day, preferably ranges from approximately 5×10⁴ toapproximately 2.5×10⁵ viable bacteria or spores per day, and morepreferably ranges from approximately 1.5×10⁵ to approximately 2×10⁵viable bacteria or spores per day.
 32. The therapeutic composition ofclaim 25, wherein said therapeutic composition additionally contains oneor more bifidogenic factors, wherein said bifidogenic oligosaccharide isselected from the group consisting of fructo-oligosaccharide (FOS),gluco-oligosaccharide (GOS), raffinose, and long-chain oligosaccharides.33. The therapeutic composition of claim 25, wherein said therapeuticcomposition additionally contains one or more bifidogenic factors andwherein said bifidogenic factor is a fructo-oligosaccharide (FOS), andwherein the total administered concentration of the bifidogenic factorwithin said therapeutic composition ranges ranging from approximately 10milligrams to approximately 20 grams per gram of therapeutic compositionper day, preferably from approximately 50 milligrams to approximately 10grams per gram of therapeutic composition per day, and more preferablyfrom approximately from approximately 150 milligrams to approximately 5grams per gram of therapeutic composition per day.
 34. The therapeuticcomposition of claim 25, wherein said pathogenic bacteria areantibiotic-resistant pathogenic bacteria.
 35. The therapeuticcomposition of claim 25, wherein the physiological location of theadministration of said therapeutic composition is selected from a groupcomprising: buccal; topical; vaginal; nasal; ocular; and oticadministration locations.
 36. The therapeutic composition of claim 25,wherein said therapeutic composition additionally comprises ananti-microbial agent which is selected from the group comprising:antibiotics, anti-fungal agents, anti-viral agents, and anti-yeastagents.
 37. The therapeutic composition of claim 25, wherein saidtherapeutic composition is additionally comprised of atherapeutically-effective concentration of an antibiotic which isselected from a group consisting of: Gentamicin; Vancomycin; Oxacillin;Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-Sulfamethoxasole; a member of the Cephlosporin antibioticfamily; a member of the Penicillin antibiotic family; a member of theFluoroquinolone antibiotic family; and a member of the Macrolideantibiotic family.
 38. The therapeutic composition of claim 25, whereinsaid therapeutic composition is additionally comprised of atherapeutically-effective concentration of an anti-fungal agent which isselected from a group consisting of: Clotrimazole, Fluconazole,Itraconazole, Ketoconazole, Miconazole, Nystatin, Terbinafine,Terconazole, and Tioconazole.
 39. A method for the treatment orprevention of a gastrointestinal infection caused by a pathogenicbacteria, comprising the administration of a therapeutically-effectiveconcentration of one or more species or strains of lactic acid-producingbacteria within a pharmaceutically-acceptable carrier suitable foradministration to the gastrointestinal tract of a vertebrate, whereinsaid lactic acid-producing bacteria possesses the ability to reduce boththe colonization rate and the potential physiologically deleteriouseffects due to the colonization of said pathogenic bacteria.
 40. Themethod of claim 39, wherein one of said lactic acid-producing bacteriais a Bacillus species which is selected from a group comprising:Bacillus coagulans; Bacillus coagulans Hammer; and Bacillus brevissubspecies coagulans, and any genetic variants thereof.
 41. The methodof claim 39, wherein one of said lactic acid-producing bacteria is aLactobacillus species which is selected from a group comprising:Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus DDS-1,Lactobacillus GG, Lactobacillus rhamnosus, Lactobacillus plantarum,Lactobacillus reuteri, Lactobacillus gasserii, Lactobacillus jensenii,Lactobacillus delbruekii, Lactobacillus, bulgaricus, Lactobacillussalivarius and Lactobacillus sporogenes (also designated as Bacilluscoagulans), and any genetic variants thereof.
 42. The method of claim39, wherein one of said lactic acid-producing bacteria is aSporolactobacillus species which is selected from a group comprising:Sporolactobacillus P44, and any genetic variants thereof.
 43. The methodof claim 39, wherein one of said lactic acid-producing bacteria is aBifidiobacterium species which is selected from a group comprising:Bifidiobacterium adolescentis, Bifidiobacterium animalis,Bifidiobacterium bifidum, Bifidiobacterium bifidus, Bifidiobacteriumbreve, Bifidiobacterium infantis, Bifidiobacterium infantis,Bifidiobacterium longum, and any genetic variants thereof.
 44. A methodfor the treatment or prevention of a gastrointestinal infection causedby a pathogenic bacteria, comprising the administration of atherapeutically-effective concentration of an anti-microbial agentselected from the group comprising an antibiotic, an anti-fungal agent,and anti-yeast agent, or an anti-viral agent, and of one or more speciesor strains of lactic acid-producing bacteria within apharmaceutically-acceptable carrier suitable for administration to thegastrointestinal tract of a vertebrate, wherein said lacticacid-producing bacteria possesses the ability to reduce both thecolonization rate and the potential physiologically deleterious effectsdue to the colonization of said pathogenic bacteria.
 45. The method ofclaim 44, wherein one of said lactic acid-producing bacteria is aBacillus species which is selected from a group comprising: Bacilluscoagulans; Bacillus coagulans Hammer; and Bacillus brevis subspeciescoagulans, and any genetic variants thereof.
 46. The method of claim 44,wherein one of said lactic acid-producing bacteria is a Lactobacillusspecies which is selected from a group comprising: Lactobacillusacidophilus, Lactobacillus casei, Lactobacillus DDS-1, Lactobacillus GG,Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri,Lactobacillus gasserii, Lactobacillus jensenii, Lactobacillusdelbruekii, Lactobacillus, bulgaricus, Lactobacillus salivarius andLactobacillus sporogenes (also designated as Bacillus coagulans), andany genetic variants thereof.
 47. The method of claim 44, wherein one ofsaid lactic acid-producing bacteria is a Sporolactobacillus specieswhich is selected from a group comprising: Sporolactobacillus P44, andany genetic variants thereof.
 48. The method of claim 44, wherein one ofsaid lactic acid-producing bacteria is a Bifidiobacterium species whichis selected from a group comprising: Bifidiobacterium adolescentis,Bifidiobacterium animalis, Bifidiobacterium bifidum, Bifidiobacteriumbifidus, Bifidiobacterium breve, Bifidiobacterium infantis,Bifidiobacterium infantis, Bifidiobacterium longum, and any geneticvariants thereof.
 49. A method for the treatment or prevention of agastrointestinal infection caused by a pathogenic bacteria, comprisingthe administration of a therapeutically-effective concentration of ananti-microbial agent, and one or more species or strains of Bacilluscoagulans within a pharmaceutically-acceptable carrier suitable foradministration to the gastrointestinal tract of a vertebrate, whereinsaid Bacillus coagulans bacterial strains possess antibiotic resistanceor decreased antibiotic sensitivity to the antibiotic selected for usein said composition and wherein said Bacillus coagulans possesses theability to reduce both the colonization rate and the potentialphysiologically deleterious effects due to the colonization of saidpathogenic bacteria.
 50. The method of claim 49, wherein said Bacilluscoagulans bacterial stain is included in said composition in a formselected from a group consisting of a dried bacterial cell mass within aflowable concentrate, a stabilized gel, or a stabilized paste.
 51. Themethod of claim 49, wherein said Bacillus coagulans bacterial stain isin form of a dried bacterial spore mass which possess the ability togerminate following administration within a flowable concentrate, astabilized gel, or a stabilized paste.
 52. The method of claim 49,wherein the total administered concentration of said therapeuticcomposition ranges from approximately 10 milligrams to approximately 10grams of composition per day, and preferably ranges from approximately0.1 grams to approximately 5 grams of composition per day.
 53. Themethod of claim 49, wherein the total administered concentration ofBacillus coagulans within said therapeutic composition ranges fromapproximately 1×10³ to approximately 1×10¹⁴ viable bacteria or sporesper day, preferably ranges from approximately 1×10⁵ to approximately1×10¹⁰ viable bacteria or spores per day, and more preferably rangesfrom approximately 5×10⁸ to approximately 1×10⁹ viable bacteria orspores per day.
 54. The method of claim 49, wherein the infection to betreated is within an adult and is caused by an antibiotic-resistantpathogen, and wherein the total administered concentration of Bacilluscoagulans within said therapeutic composition ranges from approximately1×10² to approximately 1×10⁴ viable bacteria or spores per day,preferably ranges from approximately 1×10⁸ to approximately 1×10¹⁰viable bacteria or spores per day, and more preferably ranges fromapproximately 2.5×10⁸ to approximately 1×10¹⁰ viable bacteria or sporesper day.
 55. The method of claim 49, wherein the infection to be treatedis Sudden Infant Death Syndrome (SIDS) in an infant over six months ofage and is caused by an antibiotic-resistant pathogen, and wherein thetotal administered concentration of Bacillus coagulans within saidtherapeutic composition ranges from approximately 1×10⁶ to approximately1×10⁹ viable bacteria or spores per day, preferably ranges fromapproximately 5×10⁴ to approximately 2.5×10⁵ viable bacteria or sporesper day, and more preferably ranges from approximately 1.5×10⁵ toapproximately 2×10⁵ viable bacteria or spores per day.
 56. The method ofclaim 49, wherein said therapeutic composition additionally contains oneor more bifidogenic factors, wherein said bifidogenic oligosaccharide isselected from the group consisting of fructo-oligosaccharide (FOS),gluco-oligosaccharide (GOS), raffinose, and long-chain oligosaccharides.57. The method of claim 49, wherein said therapeutic compositionadditionally contains one or more bifidogenic factors and wherein saidbifidogenic factor is a fructo-oligosaccharide (FOS), and wherein thetotal administered concentration of the bifidogenic factor within saidtherapeutic composition ranges ranging from approximately 10 milligramsto approximately 20 grams per gram of therapeutic composition per day,preferably from approximately 50 milligrams to approximately 10 gramsper gram of therapeutic composition per day, and more preferably fromapproximately from approximately 150 milligrams to approximately 5 gramsper gram of therapeutic composition per day.
 58. The method of claim 49,wherein said pathogenic bacteria are antibiotic-resistant pathogenicbacteria.
 59. The method of claim 49, wherein the physiological locationof the administration of said therapeutic composition is selected from agroup comprising: buccal; topical; vaginal; nasal; ocular; and oticadministration locations.
 60. The method of claim 49, wherein saidanti-microbial agent is selected from the group comprising: antibiotics,anti-fungal agents, anti-viral agents, and anti-yeast agents.
 61. Themethod of claim 49, wherein said anti-microbial agent is comprised of atherapeutically-effective concentration of an antibiotic selected from agroup consisting of: Gentamicin; Vancomycin; Oxacillin; Tetracyclines;Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-Sulfamethoxasole; a member of the Cephlosporin antibioticfamily; a member of the Penicillin antibiotic family; a member of theFluoroquinolone antibiotic family; and a member of the Macrolideantibiotic family.
 62. The method of claim 49, wherein saidanti-microbial agent is comprised of a therapeutically-effectiveconcentration of an anti-fungal agent selected from a group consistingof: Clotrimazole, Fluconazole, Itraconazole, Ketoconazole, Miconazole,Nystatin, Terbinafine, Terconazole, and Tioconazole.
 63. A method forthe treatment or prevention of a gastrointestinal infection caused by apathogenic bacteria, comprising the administration of atherapeutically-effective of one or more species or strains of Bacilluscoagulans within a pharmaceutically-acceptable carrier suitable foradministration to the gastrointestinal tract of a vertebrate, whereinsaid Bacillus coagulans bacterial strains possess the ability to reduceboth the colonization rate and the potential physiologically deleteriouseffects due to the colonization of said pathogenic bacteria.
 64. Themethod of claim 63, wherein said Bacillus coagulans bacterial stain isincluded in said composition in a form selected from a group consistingof a dried bacterial cell mass within a flowable concentrate, astabilized gel, or a stabilized paste.
 65. The method of claim 63,wherein said Bacillus coagulans bacterial stain is in form of a driedbacterial spore mass which possess the ability to germinate followingadministration within a flowable concentrate, a stabilized gel, or astabilized paste.
 66. The method of claim 63, wherein the totaladministered concentration of said therapeutic composition ranges fromapproximately 10 milligrams to approximately 10 grams of composition perday, and preferably ranges from approximately 0.1 grams to approximately5 grams of composition per day.
 67. The method of claim 63, wherein thetotal administered concentration of Bacillus coagulans within saidtherapeutic composition ranges from approximately 1×10³ to approximately1×10¹⁴ viable bacteria or spores per day, preferably ranges fromapproximately 1×10⁵ to approximately 1×10¹⁰ viable bacteria or sporesper day, and more preferably ranges from approximately 5×10⁸ toapproximately 1×10⁹ viable bacteria or spores per day.
 68. The method ofclaim 63, wherein the infection to be treated is within an adult and iscaused by an antibiotic-resistant pathogen, and wherein the totaladministered concentration of Bacillus coagulans within said therapeuticcomposition ranges from approximately 1×10² to approximately 1×10¹⁴viable bacteria or spores per day, preferably ranges from approximately1×10⁸ to approximately 1×10¹⁰ viable bacteria or spores per day, andmore preferably ranges from approximately 2.5×10⁸ to approximately1×10¹⁰ viable bacteria or spores per day.
 69. The method of claim 63,wherein the infection to be treated is Sudden Infant Death Syndrome(SIDS) in an infant over six months of age and is caused by anantibiotic-resistant pathogen, and wherein the total administeredconcentration of Bacillus coagulans within said therapeutic compositionranges from approximately 1×10⁶ to approximately 1×10⁹ viable bacteriaor spores per day, preferably ranges from approximately 5×10⁴ toapproximately 2.5×10⁵ viable bacteria or spores per day, and morepreferably ranges from approximately 1.5×10⁵ to approximately 2×10⁵viable bacteria or spores per day.
 70. The method of claim 63, whereinsaid therapeutic composition additionally contains one or morebifidogenic factors, wherein said bifidogenic oligosaccharide isselected from the group consisting of fructo-oligosaccharide (FOS),gluco-oligosaccharide (GOS), raffinose, and long-chain oligosaccharides.71. The method of claim 63, wherein said therapeutic compositionadditionally contains one or more bifidogenic factors and wherein saidbifidogenic factor is a fructo-oligosaccharide (FOS), and wherein thetotal administered concentration of the bifidogenic factor within saidtherapeutic composition ranges ranging from approximately 10 milligramsto approximately 20 grams per gram of therapeutic composition per day,preferably from approximately 50 milligrams to approximately 10 gramsper gram of therapeutic composition per day, and more preferably fromapproximately from approximately 150 milligrams to approximately 5 gramsper gram of therapeutic composition per day.
 72. The method of claim 63,wherein said pathogenic bacteria are antibiotic-resistant pathogenicbacteria.
 73. The method of claim 63, wherein the physiological locationof the administration of said therapeutic composition is selected from agroup comprising: buccal; topical; vaginal; nasal; ocular; and oticadministration locations.
 74. The method of claim 63, wherein saidtherapeutic composition additionally comprises an anti-microbial agentwhich is selected from the group comprising: antibiotics, anti-fungalagents, anti-viral agents, and anti-yeast agents.
 75. The method ofclaim 63, wherein said therapeutic composition is additionally comprisedof a therapeutically-effective concentration of an antibiotic which isselected from a group consisting of: Gentamicin; Vancomycin; Oxacillin;Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-Sulfamethoxasole; a member of the Cephlosporin antibioticfamily; a member of the Penicillin antibiotic family; a member of theFluoroquinolone antibiotic family; and a member of the Macrolideantibiotic family.
 76. The method of claim 63, wherein said therapeuticcomposition is additionally comprised of a therapeutically-effectiveconcentration of an anti-fungal agent which is selected from a groupconsisting of: Clotrimazole, Fluconazole, Itraconazole, Ketoconazole,Miconazole, Nystatin, Terbinafine, Terconazole, and Tioconazole.
 77. Atherapeutic composition for the treatment or prevention of agastrointestinal infection caused by a pathogenic bacteria, comprising atherapeutically-effective concentration of an antibiotic and one or moreBacillus coagulans bacterial strains which possess antibiotic resistanceor decreased antibiotic sensitivity to the antibiotic selected for usein said composition, within a pharmaceutically-acceptable carriersuitable for administration to the gastrointestinal tract of avertebrate, and wherein said Bacillus coagulans bacterial strain is inthe form of spores which possess the ability to germinate followingadministration.
 78. A therapeutic composition for the treatment orprevention of a gastrointestinal infection caused by a pathogenicbacteria, comprising a therapeutically-effective concentration of ananti-fungal agent and one or more Bacillus coagulans bacterial strainswhich possess resistance or decreased sensitivity to the anti-fungalagent selected for use in said composition, within apharmaceutically-acceptable carrier suitable for administration to thegastrointestinal tract of a vertebrate, and wherein said Bacilluscoagulans bacterial strain is in the form of spores which possess theability to germinate following administration.
 79. A therapeuticcomposition for the treatment or prevention of a gastrointestinalinfection caused by a pathogenic bacteria, comprising atherapeutically-effective concentration of an anti-yeast agent and oneor more Bacillus coagulans bacterial strains which possess resistance ordecreased sensitivity to the anti-yeast agent selected for use in saidcomposition, within a pharmaceutically-acceptable carrier suitable foradministration to the gastrointestinal tract of a vertebrate, andwherein said Bacillus coagulans bacterial strain is in the form ofspores which possess the ability to germinate following administration.80. A therapeutic composition for the treatment or prevention of agastrointestinal infection caused by a pathogenic bacteria, comprising atherapeutically-effective concentration of an anti-viral agent and oneor more Bacillus coagulans bacterial strains which possess resistance ordecreased sensitivity to the anti-viral agent selected for use in saidcomposition, within a pharmaceutically-acceptable carrier suitable foradministration to the gastrointestinal tract of a vertebrate, andwherein said Bacillus coagulans bacterial strain is in the form ofspores which possess the ability to germinate following administration.81. A method for the treatment or prevention of a gastrointestinalinfection caused by a pathogenic bacteria, comprising the administrationof a therapeutically-effective concentration of an antibiotic and one ormore Bacillus coagulans bacterial strains which possess antibioticresistance or decreased antibiotic sensitivity to the antibioticselected for use in said composition, within apharmaceutically-acceptable carrier suitable for administration to thegastrointestinal tract of a vertebrate, and wherein said Bacilluscoagulans bacterial strain is in the form of spores which possess theability to germinate following administration.
 82. A method for thetreatment or prevention of a gastrointestinal infection caused by apathogenic bacteria, comprising the administration of atherapeutically-effective concentration of an anti-fungal agent and oneor more Bacillus coagulans bacterial strains which possess resistance ordecreased sensitivity to the anti-fungal agent selected for use in saidcomposition, within a pharmaceutically-acceptable carrier suitable foradministration to the gastrointestinal tract of a vertebrate, andwherein said Bacillus coagulans bacterial strain is in the form ofspores which possess the ability to germinate following administration.83. A method for the treatment or prevention of a gastrointestinalinfection caused by a pathogenic bacteria, comprising the administrationof a therapeutically-effective concentration of an anti-yeast agent andone or more Bacillus coagulans bacterial strains which possessresistance or decreased sensitivity to the anti-yeast agent selected foruse in said composition, within a pharmaceutically-acceptable carriersuitable for administration to the gastrointestinal tract of avertebrate, and wherein said Bacillus coagulans bacterial strain is inthe form of spores which possess the ability to germinate followingadministration.
 84. A method for the treatment or prevention of agastrointestinal infection caused by a pathogenic bacteria, comprisingthe administration of a therapeutically-effective concentration of ananti-viral agent and one or more Bacillus coagulans bacterial strainswhich possess resistance or decreased sensitivity to the anti-viralagent selected for use in said composition, within apharmaceutically-acceptable carrier suitable for administration to thegastrointestinal tract of a vertebrate, and wherein said Bacilluscoagulans bacterial strain is in the form of spores which possess theability to germinate following administration.
 85. A composition forestablishing or re-establishing the microbial flora of a vertebrategastrointestinal tract concomitant with, or following the previousadministration of an anti-microbial agent which is selected from thegroup comprising: antibiotics, anti-fungal agents, anti-yeast agents,and anti-viral agents, said composition comprising of one or moreBacillus coagulans bacterial strains and one or more bifidogenic factorswithin a pharmaceutically-acceptable carrier suitable for oraladministration to the gastrointestinal tract of a vertebrate, whereinsaid Bacillus coagulans bacterial strains possess resistance ordecreased sensitivity to said anti-microbial agents.
 86. A method forestablishing or re-establishing the microbial flora of a vertebrategastrointestinal tract concomitant with, or following the previousadministration of an antimicrobial agent which is selected from thegroup comprising: antibiotics, anti-fungal agents, anti-yeast agents,and anti-viral agents, comprising the administration of a compositioncomprising of one or more Bacillus coagulans bacterial strains and oneor more bifidogenic factors within a pharmaceutically-acceptable carriersuitable for oral administration to the gastrointestinal tract of avertebrate, wherein said Bacillus coagulans bacterial strains possessresistance or decreased sensitivity to said anti-microbial agents.
 87. Amethod for producing resistance or decreased sensitivity to ananti-microbial agent in a Bacillus coagulans bacterial straincomprising: (i) culturing said Bacillus coagulans bacterial strain on aPetri dish containing an agar-based medium capable of supporting growthof said bacterial strain and a sub-lethal concentration of a selectedanti-microbial agent; (ii) viable colonies are thenindividually-selected and re-cultured on a Petri dish containing anagar-based medium capable of supporting growth of said bacterial strainand an increased concentration of the selected anti-microbial agentwhich is still sub-lethal to said bacterial strain; and (iii) repeatingstep (ii) utilizing gradually increasing, sub-lethal concentrations ofthe selected anti-microbial agent until such time as said isolatedBacillus coagulans bacterial strain acquires the ability to remainviable in concentrations of the selected anti-microbial agent whichapproximate those therapeutically-effective concentrations of saidanti-microbial agent which will be utilized in the concomitantlyadministered composition.
 88. The method of claim 87, wherein saidanti-microbial agent is an antibiotic which is selected from a groupconsisting of: Gentamicin; Vancomycin; Oxacillin; Tetracyclines;Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-sulfamethoxasole; a member of the Cephlosporin antibioticfamily; a member of the Penicillin antibiotic family; a member of theFluoroquinolone antibiotic family; and a member of the Macrolideantibiotic family.
 89. The method of claim 87, wherein saidanti-microbial agent is an anti-fungal agent which is selected from agroup consisting of: Clotrimazole, Fluconazole, Itraconazole,Ketoconazole, Miconazole, Nystatin, Terbinafine, Terconazole, andTioconazole.
 90. A method for producing antibiotic-resistance ordecreased antibiotic sensitivity in an isolated Bacillus coagulansbacterial strain comprising the utilization of conjugative transposons,possessing DNA sequences which are capable of conferring resistance tothe selected antibiotic, which allow the direct transfer of saidantibiotic-resistance genes without a plasmid intermediary.
 91. A methodfor producing antibiotic-resistance or decreased antibiotic sensitivityin an isolated Bacillus coagulans bacterial strain comprising theutilization of recombinant DNA-based techniques to produce a plasmidwhich possesses DNA sequences which are capable of conferring resistanceto the selected antibiotic and transfecting the Bacillus coagulansbacterial strain with said plasmid.
 92. A probiotic compositioncomprising one or more Bacillus coagulans bacterial strains within apharmaceutically-acceptable carrier suitable for oral administration tothe gastrointestinal tract of a vertebrate, wherein said Bacilluscoagulans bacterial strain is capable of growing at a temperature ofapproximately 30° C. to approximately 65° C., produces L(+)dextrorotatory lactic acid, produces spores resistant to temperatures ofup to approximately 90° C., and exhibits probiotic activity whichinhibits the growth of bacteria, yeast, fungi, virus, or anycombinations thereof.
 93. The probiotic composition of claim 92, whereinsaid probiotic activity results from the vegetative growth of theisolated Bacillus species bacterial strain.
 94. The probioticcomposition of claim 92, wherein said probiotic activity results from anextracellular product produced by the isolated Bacillus speciesbacterial strain.
 95. A probiotic composition comprising an antibioticand a one or more Bacillus coagulans bacterial strains which possessesantibiotic resistance or decreased antibiotic sensitivity to theantibiotic selected for use in said composition, within apharmaceutically-acceptable carrier suitable for oral administration tothe gastrointestinal tract of a vertebrate, wherein said Bacilluscoagulans bacterial strain is capable of growing at a temperature ofapproximately 30° C. to approximately 65° C., produces L(+)dextrorotatory lactic acid, produces spores resistant to temperatures ofup to approximately 90° C., and exhibits probiotic activity whichinhibits the growth of bacteria, yeast, fungi, virus, or anycombinations thereof.
 96. The probiotic composition of claim 95, whereinsaid probiotic activity results from both the antibiotic and thevegetative growth of the isolated Bacillus species bacterial strain. 97.The probiotic composition of claim 95, wherein said probiotic activityresults from both the antibiotic and an extracellular product producedby the isolated Bacillus species bacterial strain.
 98. A composition forthe treatment or prevention of a gastrointestinal infection caused by apathogenic bacteria, comprising a therapeutically-effectiveconcentration of an extracellular product of a Bacillus coagulans strainin a pharmaceutically-acceptable carrier which is suitable for oraladministration to the gastrointestinal tract of a vertebrate.
 99. Amethod for the treatment or prevention of a gastrointestinal infectioncaused by a pathogenic bacteria, comprising the administration of atherapeutically-effective concentration of an extracellular product of aBacillus coagulans strain in a pharmaceutically-acceptable carrier whichis suitable for oral administration to the gastrointestinal tract of avertebrate.
 100. A composition for the treatment or prevention of agastrointestinal infection caused by a pathogenic bacteria, comprising atherapeutically-effective concentration of an antimicrobial agentselected from the group comprising: an antibiotic, an anti-fungal agent,an anti-yeast agent, or an anti-viral agent, and extracellular productof a Bacillus coagulans strain in a pharmaceutically-acceptable carrierwhich is suitable for oral administration to the gastrointestinal tractof a vertebrate.
 101. A method for the treatment or prevention of agastrointestinal infection caused by a pathogenic bacteria, comprisingthe administration of a therapeutically-effective concentration of ananti-microbial agent selected from the group comprising: an antibiotic,an anti-fungal agent, an anti-yeast agent, or an anti-viral agent, of anextracellular product of a Bacillus coagulans strain in apharmaceutically-acceptable carrier which is suitable for oraladministration to the gastrointestinal tract of a vertebrate.
 102. Acomposition for inhibiting parasite growth in the gastrointestinal tractand/or feces of animals comprising an effective amount of diatomaceousearth and a one or more viable, non-pathogenic lactic acid bacteria.103. The composition of claim 102, wherein said diatomaceous earth is atleast 80% (w/w) Melosira.
 104. The composition of claim 102, whereinsaid diatomaceous earth is present in an amount of from approximately0.1 to 95 weight percent.
 105. The composition of claim 102, whereinsaid diatomaceous earth is present in an amount of from about 1 to 10weight percent.
 106. The composition of claim 102 wherein saidnon-pathogenic lactic acid bacteria is selected from the groupconsisting of Lactobacillus acidophilus, Lactobacillus salivarius,Lactobacillus planterum, Lactobacillus deibrukeil, Lactobacillussporegenes, Sporolactobacillus P44, Lactobacillus rhainnosus,Lactobacillus casei, Lactobacillus gaseril, Lactobacillus jensenhi,Lactobacillus salivarius, Lactobacillus rhamnosus CC, Lactobacillusbulgaricus, Lactobacillus thermophilus, Lactobacillus fermenturn,Lactobacillus cereale, Bifidobacterium longum, Bifidiobacterium bitidum,Bifidiobacterium intantus, Bifidiobacterium bitidus, Bifidiobacteriuminfantis, Bifidiobacterium ion gum, Bifidiobacterium breve,Bifidiobacterium animalis, Bifidiobacterium adolescentis, Bacilluscoagulans, Bacillus laevolacticus, and Bacillus brevis subsp. coagulans.107. The composition of claim 102, wherein said Bacillus coagulans isBacillus coagulans subspecies Hammer.
 108. The composition of claim 102,wherein the said bacteria is included in the composition in the form ofspores.
 109. The composition of claim 102, wherein said bacteria isincluded in the composition in the form of a dried cell mass.
 110. Thecomposition of claim 102, wherein said composition is formulated into aliquid, paste, powder, granules, pellets or tablets.
 111. Thecomposition of claim 102, wherein said composition contains 1×10³ to1×10¹⁴ CFU of viable bacteria or spores per gram of composition. 112 Thecomposition of claim 102, wherein said composition further comprises aneffective amount of a bifidogenic oligosaccharide.
 113. The compositionof claim 112, wherein said bifidogenic oligosaccharide is selected fromthe group consisting of fructo-oligosaccharide (FOS),gluco-oligosaccharide (GOS), raffinose, and long-chain oligosaccharides.114. The composition of claim 112, wherein said oligosaccharidecomprises polymers of having a polymer chain length of from about 4 to100 sugar units.
 115. The composition of claim 113, wherein saidcomposition comprises about 10 milligrams to about 1 gram of FOS pergram of composition.
 116. The composition of claim 113, wherein saidcomposition comprises from 100 to 500 milligrams of FOS per grain ofcomposition.
 117. The composition of claim 102 further comprising anutritional supplement selected from the group consisting of vitaminsand minerals.
 118. The composition of claim 117, wherein said vitaminsare selected from the group consisting of Vitamin A, C, E, C, B-1, B-2,B-3, B-6, B-12, K and pantothenic acid.
 119. The composition of claim117, wherein said minerals are selected from the group consisting ofcalcium, magnesium, phosphorus, zinc, manganese, copper and potassium.120. The composition of claim 117, wherein said minerals are selectedfrom the group consisting of antimony, barium, berylium, bismuth, boron,bromine, chromium, cobalt, germanium, gold, iodine, iron, lithium,nickel, palladium, platinum, selenium, silicon, silver, strontium, tin,titanium, tungsten, vanadium and zirconium.
 121. The composition ofclaim 102, wherein said composition further comprises a food substanceor flavoring.
 122. The composition of claim 102, wherein the compositionfurther comprises powdered food supplement, a packaged food, a liquidformula or an oral electrolyte maintenance formulation.
 123. Thecomposition of claim 122, wherein said oral electrolyte maintenanceformulation is a powder comprising sodium chloride, potassium citrate,citric acid or glucose.
 124. A method for inhibiting parasite infectionsin the gastro-intestinal tract of an animal comprising administering tothe digestive tract of an animal a composition according to claim 102.125. The method of claim 124, wherein said administering comprises oralingestion of said composition.
 126. The method of claim 124, whereinsaid administering comprises introducing into the digestive tract from0.1 to 50 grams of said composition per 100 kilogram animal body weightper day.
 127. The method of claim 124, wherein said administeringcomprises introducing into the digestive tract from approximately 1×10²to approximately 1×10¹⁰ viable bacteria or spores per 100 kilogramanimal body weight per day.
 128. The method of claim 124, wherein saidadministering comprises introducing into the digestive tract from 1×10³to 1×10⁶ viable bacteria or spores per 100 kilogram animal body weightper day.
 129. The method of claim 124, wherein said administeringcomprises introducing into the digestive tract from 1×10⁶ to 1×10⁹viable bacteria or spores per 100 kilogram animal body weight per day.130. The method of claim 124, wherein said administering comprisesintroducing into the digestive tract from 50 milligrams to 10 gramsdiatomaceous earth per kilogram of animal body weight per day.
 131. Themethod of claim 124, wherein said administering comprises introducinginto the digestive tract from 200 to 500 milligrams diatomaceous earthper kilogram of animal body weight per day.
 132. The method of claim124, wherein said composition further comprises an effective amount of abifidogenic oligosaccharide.
 133. The method of claim 132, wherein saidadministering comprises introducing into the digestive tract from 10milligrams to 20 grams of bifidogenic oligosaccharide per 100 kilogramanimal body weight per day.
 134. The method of claim 132, wherein saidadministering comprises introducing into the digestive tract from 150milligrams to 5 grams of bifidogenic oligosaccharide per 100 kilogramanimal body weight per day.
 135. A method for reducing insectpopulations in animal manure comprising administering to the digestivetract of an animal a composition according to claim
 102. 136. The methodof claim 135, wherein said administering comprises oral ingestion ofsaid composition.
 137. The method of claim 135, wherein saidadministering comprises introducing into the digestive tract from 0.1 to50 grams of said composition per 100 kilogram animal body per day. 138.The method of claim 135, wherein said administering comprisesintroducing into the digestive tract from 1×10² to 1×10¹⁰ viablebacteria or spores per 100 kilogram animal body weight per day.
 139. Themethod of claim 135, wherein said administering comprises introducinginto the digestive tract from 1×10³ to 1×10⁶ viable bacteria or sporesper 100 kilogram animal body weight per day.
 140. The method of claim135, wherein said administering comprises introducing into the digestivetract from 1×10⁶ to 1×10⁹ viable bacteria or spores per 100 kilogramanimal body weight per day.
 141. The method of claim 135, wherein saidadministering comprises introducing into the digestive tract from 50milligrams to 10 grams diatomaceous earth per kilogram of animal bodyweight per day.
 142. The method of claim 135, wherein said administeringcomprises introducing into the digestive tract from 200 to 500milligrams diatomaceous earth per kilogram of animal body weight perday.
 143. The method of claim 135, wherein said composition furthercomprises an effective amount of a bifidogenic oligosaccharide.
 144. Themethod of claim 143, wherein said administering comprises introducinginto the digestive tract from 10 milligrams to 20 grams of bifidogenicoligosaccharide per 100 kilogram animal body weight per day.
 145. Themethod of claim 143, wherein said administering comprises introducinginto the digestive tract from 150 milligrams to 5 grams of bifidogenicoligosaccharide per 100 kilogram animal body weight per day.
 146. Asystem for inhibiting parasite infections in the gastro-intestinal tractof an animal comprising a container having a label and a compositionaccording to claim 102, wherein said label comprises instructions foruse of said composition for inhibiting parasite infections in thegastro-intestinal tract of an animal.
 147. A system for reducing insectpopulations in animal manure comprising a container having a label and acomposition according to claim 102, wherein said label comprisesinstructions for use of said composition for reducing insect populationsin animal manure.
 148. A composition for inhibiting parasite growth inthe gastro-intestinal tract and/or feces of animals comprising aneffective amount of diatomaceous earth wherein said diatomaceous earthin said composition consists essentially of at least 50% (w/w) Melosira.149. The composition of claim 148, wherein said diatomaceous earthconsists essentially of at least 80% (w/w.) Melosira.
 150. Thecomposition of claim 148, wherein said composition further comprises aculture of viable non-pathogenic lactic acid-producing bacteria.
 151. Amethod for inhibiting parasite infections in the gastrointestinal tractof an animal comprising administering to the digestive tract of ananimal a composition according to claim
 148. 152. A method for reducinginsect populations in animal manure comprising administering to thedigestive tract of an animal mammal a composition according to claim148.
 153. A system for inhibiting parasite infections in thegastro-intestinal tract of an animal comprising a container having alabel and a composition according to claim 148, wherein said labelcomprises instructions for use of said composition for inhibitingparasite infections in the gastro-intestinal tract of an animal.
 154. Asystem for reducing insect populations in animal manure comprising acontainer having a label and a composition according to claim 148,wherein said label comprises instructions for use of said compositionfor reducing insect populations in animal manure.